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Mass spectrometry versus western blotting for validation

Mass spectrometry versus western blotting for validation


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I have mass spectrometry data (LC-MS/MS) from rat cortices under either drug or control treatments. The results were performed in triplicate (three pairs of rats, drug or control per pair). In addition to some of the bioinformatics analysis I am doing, I will have to validate some of the fold-changes from the mass spectrometry by Western blot in order to have my results published.

Some of the proteins I have chosen to to validate work very nicely by Western blot which captures the same trend as the mass spectrometry. However, other proteins that I have attempted to validate go in the exact opposite direction.

My questions:

  1. In general, how accurate is mass spectrometry compared to Western blotting?
  2. Is it unusual to have a lot of variability between the results of the two methods? I am worried that I might have to try multiple proteins before I see the results replicated and this seems problematic to me. That leads me to the third question.
  3. Which result should I trust?

Other information - Some of the proteins I have chosen for validation come from lower in the list where not as many peptide fragments were detected. In some cases, fragments were detected in only 2 of the 3 biological replicates. I did not chose to validate proteins where the 2 or 3 replicates varied widely in normalized spectral count values so I would expect these readings to be accurate. Additionally, the mass spectrometry part of the experiment was performed by a reputable lab that has filtered erroneous data using FDR cutoffs. There is a high correlation overall between all three replicates for a particular treatment and low standard error between samples across the population so, statistically, I trust the mass spec results.

If you need more information please ask - I don't know what exactly will be useful to answer my questions.

EDIT :

This article discusses the justification for using western blotting to validate mass spec results. It suggests using selected reaction monitoring assays as an alternative. Some of the points made by commenters in this thread are reiterated in this article.


It is a common practice to prove a result using an orthogonal technique. Like RNAseq followed by qRT-PCR etc.

Western blotting is not a robust technique and cross comparisons are difficult because of difference in the avidities/affinities of different antibodies. So comparisons can be made only with one protein-control pair in different conditions.

LC-MS is more sensitive and less biased(IMO). So the general trick is- don't do westerns for all proteins just report the ones that behave well (say that you "chose" these because they are important). I know this is a wrong practice and I should not be advocating it. For your own scientific validation try it with another MS technique; if you used ESI-Quadrupole/ion-Trap etc then try with MALDI-TOF or iTRAQ. Some luddites will continue to cling to westerns.

I think it is better to not test the proteins that have low peptide counts. See what is known as MA plot. This is frequently used for microarrays. HereMdenotes fold change andAdenotes total expression in both samples. Don't pick proteins that are low in expression in both samples; they might not be meaningful. For example if you have2molecules ofXand100molecules ofYin control condition and5molecules ofXand200molecules ofYin test; then the fold change inXwould seem important; however this change may not be relevant and can be a result of stochastic fluctuation/measurement error. If you have many samples you can see if something is stochastic or not but the limitation is the number of samples.

Note: I am not saying that 2→5 molecule increment should be meaningless. But to know if they have a meaning or not you would require more complex models; better avoid them at this moment.


Proteomics by mass spectrometry: approaches, advances, and applications

Mass spectrometry (MS) is the most comprehensive and versatile tool in large-scale proteomics. In this review, we dissect the overall framework of the MS experiment into its key components. We discuss the fundamentals of proteomic analyses as well as recent developments in the areas of separation methods, instrumentation, and overall experimental design. We highlight both the inherent strengths and limitations of protein MS and offer a rough guide for selecting an experimental design based on the goals of the analysis. We emphasize the versatility of the Orbitrap, a novel mass analyzer that features high resolution (up to 150,000), high mass accuracy (2-5 ppm), a mass-to-charge range of 6000, and a dynamic range greater than 10(3). High mass accuracy of the Orbitrap expands the arsenal of the data acquisition and analysis approaches compared with a low-resolution instrument. We discuss various chromatographic techniques, including multidimensional separation and ultra-performance liquid chromatography. Multidimensional protein identification technology (MudPIT) involves a continuum sample preparation, orthogonal separations, and MS and software solutions. We discuss several aspects of MudPIT applications to quantitative phosphoproteomics. MudPIT application to large-scale analysis of phosphoproteins includes (a) a fractionation procedure for motif-specific enrichment of phosphopeptides, (b) development of informatics tools for interrogation and validation of shotgun phosphopeptide data, and (c) in-depth data analysis for simultaneous determination of protein expression and phosphorylation levels, analog to western blot measurements. We illustrate MudPIT application to quantitative phosphoproteomics of the beta adrenergic pathway. We discuss several biological discoveries made via mass spectrometry pipelines with a focus on cell signaling proteomics.


Validation of protein carbonyl measurement: a multi-centre study

Protein carbonyls are widely analysed as a measure of protein oxidation. Several different methods exist for their determination. A previous study had described orders of magnitude variance that existed when protein carbonyls were analysed in a single laboratory by ELISA using different commercial kits. We have further explored the potential causes of variance in carbonyl analysis in a ring study. A soluble protein fraction was prepared from rat liver and exposed to 0, 5 and 15min of UV irradiation. Lyophilised preparations were distributed to six different laboratories that routinely undertook protein carbonyl analysis across Europe. ELISA and Western blotting techniques detected an increase in protein carbonyl formation between 0 and 5min of UV irradiation irrespective of method used. After irradiation for 15min, less oxidation was detected by half of the laboratories than after 5min irradiation. Three of the four ELISA carbonyl results fell within 95% confidence intervals. Likely errors in calculating absolute carbonyl values may be attributed to differences in standardisation. Out of up to 88 proteins identified as containing carbonyl groups after tryptic cleavage of irradiated and control liver proteins, only seven were common in all three liver preparations. Lysine and arginine residues modified by carbonyls are likely to be resistant to tryptic proteolysis. Use of a cocktail of proteases may increase the recovery of oxidised peptides. In conclusion, standardisation is critical for carbonyl analysis and heavily oxidised proteins may not be effectively analysed by any existing technique.

Keywords: Aldehyde reactive probe Carbonyl ELISA Mass spectrometry Oxidised protein Western blot Protein oxidation.

Copyright © 2015. Published by Elsevier B.V.

Figures

Primary and secondary protein carbonyls…

Primary and secondary protein carbonyls and their derivatisation by DNPH.

Calculation for carbonyl quantitation by…

Calculation for carbonyl quantitation by analysis of DNP adducts.

Semi-quantitative soluble liver protein carbonyl…

Semi-quantitative soluble liver protein carbonyl content following 0–15 min UV irradiation. (A) Coomassie…

Quantitation of the protein carbonyls…

Quantitation of the protein carbonyls in soluble protein rat liver extract. Densitometric analysis…

Protein carbonylation in rat liver…

Protein carbonylation in rat liver soluble protein fraction following increase in irradiation by…


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New Approaches to Antibody Validation Using Immunoprecipitation and Mass Spectrometry

Antibodies are used in a broad range of research and diagnostic applications for the enrichment, detection, and quantitation of proteins and their modifications. Hundreds of thousands of antibodies are commercially available against thousands of proteins and their modifications. Unfortunately, many antibodies are poorly characterized, resulting in wasted time and cost as well as potentially flawed research conclusions. To verify the performance and specificity of Thermo Scientific antibodies, we have created a comprehensive workflow to assess antibody specificity using immunoprecipitation combined with mass spectrometry (IP-MS). In preliminary experiments, we screened more than 500 antibodies to nearly 100 key cancer signaling proteins expressed across 12 cultured tumor cell lines. Approximately 70% of antibodies previously validated for immunocapture could be used to capture and identify the intended target, interacting proteins, and off-targets, and

40% of antibodies not previously validated for IP were positive by IP-MS. To demonstrate the efficacy of these antibodies, we used a set of these antibodies to simultaneously immunocapture twelve proteins in the Akt/mTOR pathway, and then quantified the proteins and their phosphorylation in four IGF-stimulated cell lines using MS-based targeted quantification. Benchmarking of these multiplexed IP-MS assays showed moderate correlation to quantitation with more traditional Western blotting, ELISA, and Luminex assays.


Abstract

Protein carbonyls are widely analysed as a measure of protein oxidation. Several different methods exist for their determination. A previous study had described orders of magnitude variance that existed when protein carbonyls were analysed in a single laboratory by ELISA using different commercial kits. We have further explored the potential causes of variance in carbonyl analysis in a ring study. A soluble protein fraction was prepared from rat liver and exposed to 0, 5 and 15 min of UV irradiation. Lyophilised preparations were distributed to six different laboratories that routinely undertook protein carbonyl analysis across Europe. ELISA and Western blotting techniques detected an increase in protein carbonyl formation between 0 and 5 min of UV irradiation irrespective of method used. After irradiation for 15 min, less oxidation was detected by half of the laboratories than after 5 min irradiation. Three of the four ELISA carbonyl results fell within 95% confidence intervals. Likely errors in calculating absolute carbonyl values may be attributed to differences in standardisation. Out of up to 88 proteins identified as containing carbonyl groups after tryptic cleavage of irradiated and control liver proteins, only seven were common in all three liver preparations. Lysine and arginine residues modified by carbonyls are likely to be resistant to tryptic proteolysis. Use of a cocktail of proteases may increase the recovery of oxidised peptides. In conclusion, standardisation is critical for carbonyl analysis and heavily oxidised proteins may not be effectively analysed by any existing technique.


Introduction

Most biological processes involve the action and regulation of multiprotein complexes. In many cases, separate properties such as subcellular localization, catalytic activity, and substrate specificity are determined by different polypeptides in a holoenzyme complex, and specific protein interaction partners may be present in nonstoichiometric amounts. For example, catalytic subunits such as protein phosphatase 1 (PP1) can interact with a spectrum of alternative protein partners, which thus bind nonstoichiometrically to generate a range of holoenzymes with different specificities (for review see Moorhead et al., 2007). This can make it difficult to distinguish specific but low abundance interacting proteins from the larger number of low affinity, but abundant, contaminant proteins that are inevitably recovered using commonly used methods such as pull-down or immunoprecipitation strategies. A key goal in most areas of cell biology, therefore, is the characterization of the protein components of multiprotein complexes through the reliable identification of specific protein interaction partners.

Any putative interaction partner identified either through affinity purification or biochemical fractionation must be validated to confirm its physiological relevance. These downstream validation experiments, involving detailed molecular characterization, are both costly and time consuming and thus it is imperative to focus resources on those subsets of potential interactions with a high probability of biological significance. Continuing improvement in the sensitivity and resolution of the mass spectrometric technology for protein identification, for example, allows for the identification of ever larger numbers of proteins in immunoaffinity and pull-down experiments. In addition to bona fide interaction partners, however, these expanding lists include increased numbers of contaminant proteins, including those that bind nonspecifically to the affinity matrix. The problem of nonspecific binding cannot be overcome satisfactorily using high stringency purification methods although this can reduce the level of nonspecific binding, it will inevitably also remove low abundance and low affinity specific partner proteins. The most effective strategy must therefore preserve all specific interaction events, which inevitably results in a large number of nonspecific proteins also copurifying that must be identified and discarded.

To solve this problem, we and others have demonstrated that a quantitative mass spectrometry–based approach combined with isotope labeling can help to distinguish which of the many proteins identified in a pull-down or immunoprecipitation experiment represent specific binding. This is done by the inclusion of a negative control, which provides a background of contaminant proteins that bind nonspecifically to the affinity matrix and/or the fusion tag, against which proteins that bind specifically to the protein of interest clearly stand out (for review see Vermeulen et al., 2008). For example, using a combination of stable isotope labeling with amino acids in cell culture (SILAC)–based quantitative proteomics (Ong et al., 2002) with immunoprecipitation of GFP-tagged fusion proteins, we revealed differences in binding partners for two different isoforms of the nuclear protein phosphatase, PP1 (Trinkle-Mulcahy et al., 2006). Other groups have used a similar approach based on tagged bait proteins to map the spectrum of human 26S proteasome interacting proteins (Wang and Huang, 2008) and to detect dynamic members of transcription factor complexes (Mousson et al., 2008). Isotope-based quantitative approaches have also been used to define tagged protein complexes in yeast (Ranish et al., 2003 Tackett et al., 2005) and both tagged and endogenous protein complexes in mammalian cells (Blagoev et al., 2003 Cristea et al., 2005 Selbach and Mann, 2006).

Although the isotope labeling strategy used in a SILAC affinity purification approach provides great help in separating specific from nonspecific interactors, experience shows that not all specific interactions can be unambiguously determined, particularly near the threshold level where signal-to-noise ratios are close to background. Here we describe a new SILAC-based mass spectrometry strategy that specifically addresses this issue, incorporating methods to increase the signal, i.e., the abundance of purified protein complexes, while reducing or filtering out the noise, i.e., proteins that bind nonspecifically to the affinity matrix, tag, and/or antibody.

The efficiency of detecting interaction partners relies upon efficient depletion of the targeted complex. Here we show that GFP-tagged proteins can be near quantitatively depleted using the recently developed GFP binder (Rothbauer et al., 2008). The GFP binder is an Escherichia coli–expressed 16-kD protein derived from a llama heavy chain antibody that binds with high affinity and specificity to GFP. This underlines the utility of using GFP as a dual tag for both affinity purification and in vivo fluorescence microscopy. Furthermore, characterizing the proteins that bind nonspecifically to three of the most commonly used affinity matrices, in either whole cell, nuclear, or cytoplasmic extracts of mammalian cells, provides a “bead proteome” filter. This facilitates distinguishing specific from nonspecific binding proteins and thereby allows objective prioritization of suitable targets for detailed molecular characterization.

In summary, we present here a powerful and reliable workflow that can be applied to analyze affinity-purified protein complexes isolated using either tagged fusion proteins or via immunoprecipitation of endogenous proteins.


Materials and methods

Biological materials

In this study, cDNA libraries, expression vectors pET-30a (Novagen) and pET30a-GST, a glutathione S-transferase (GST) tag-containing version of pET30a, created in our laboratory ( Cao et al., 2010), and bacterial strains (DH5α, BL21, and ER2566) were used. Restriction enzymes BamHI, XhoI, EcoRI, HindIII, T4 DNA ligase, and Ex Taq DNA polymerase were purchased from TAKARA.

Rice samples

Four kinds of rice samples were collected and used for western blotting analysis in this study: (i) 10 samples from the seedling (shoot and root), tillering (leaf and stem), booting (flag leaf and young panicle), flowering (flag leaf and panicle), and filling stages (flag leaf and seed) of rice variety 93-11 (Oryza sativa L.) (ii) seven leaf samples collected at 4 h intervals starting at 12 pm within a single day (iii) eight samples from leaves of the 4021-3, homozygous transgenic rice line with the bacterial blight resistance gene Xa21 ( Xiang et al., 2006), inoculated with the incompatible Philippine race 6 of Xanthomonas oryzae pv. oryzae (Xoo) at 0, 1, 2, 4, and 8 h, and 1, 3, and 5 d and (iv) four samples from wheat, maize, cotton, and A. thaliana leaves during the growing period. All materials were frozen using liquid nitrogen and stored at –70 °C until use.

Antigenic peptide prediction and primer design

BEPITOPE software ( Odorico and Pellequer, 2003) was used to predict antigenic fragments from which those which were unique in the rice genome, once verified by BLASTP, were chosen as the antigen to generate specific antibodies against target proteins. PrimerCE software ( Cao et al., 2010) was used to design the primers based on the coding sequence (CDS) downloaded from the TIGR database (http://rice.plantbiology.msu.edu/) as shown below. The underlined letters are restriction enzyme recognition sites. The detailed positions in full-length cDNA and the peptide sequences are listed in Table 1. Os09g30418.1F, 5′-CG GGATCC TTCGCCTTCCAGGCCGAGAT-3′ Os09g30418.1R, 5′-CCG CTCGAG CTCCTCAAGGTATTCCAGCTGA-3′ Os05g04510.1F, 5′-G GAATTC CTTGGCGCTCGTCTTACGGAGG-3′ Os05g04510.1R, 5′-CCC AAGCTT GCCACTAGCAACAATGCTCTTGG-3′ Os06g46770.2F, 5′-G GAATTC ATGCAGATCTTTGTGAAGACCC-3′ Os06g46770.2R, 5′-CCC AAGCTT CCTGAGCCTGAGCACAAGGTG-3′ Os03g08020.1F, 5′-G GAATTC AAGAACGTTGCGGTGAAGG-3′ Os03g08020.1R, 5′-CCC AAGCTT TCATTTCTTCTTGGCGGCAG-3′ Os03g50890.1F, 5′-G GAATTC ACCATTGGTGCTGAGCGTTTC-3′ Os03g50890.1R, 5′-CCC AAGCTT TTAGAAGCATTTCCTGTGCACAAT-3′.

Rice reference protein candidates

Gene name Locus number Annotation Mol. wt (kDa) Antigen Fragment length (position)/peptide sequence
HSP Os09g30418.1 Heat shock protein 94 Expressed protein 182 amino acids (8–189)
UBQ Os06g46770.2 Polyubiquitin containing 7 ubiquitin monomers 60 Expressed protein 150 amino acids (1–150)
TUB Os01g59150.1 Tubulin beta-4 chain 50 Synthesized peptide QYQDATADEEGEYEDEEQQ
eEF-1α Os03g08020.1 Elongation factor 1-α 49 Expressed protein 148AA(301–448)
eIF-4α Os02g05330.1 Eukaryotic initiation factor 4-α 47 Synthesized peptide DAKHYDSKMQELLHQGDNEE
SAMS Os05g04510.1 S-Adenosylmethionine synthetase 1 43 Expressed protein 150 amino acids (151–300)
ACT Os03g50890.1 Actin-1 42 Expressed protein 128 amino acids(251–378)
GAPDH Os04g40950.1 Glyceraldehyde-3-phosphate dehydrogenase, cytosolic 37 Synthesized peptide DLVSTDFQGDNRSSIFDAKAGI
UBC Os02g42314.2 Ubiquitin-conjugating enzyme E2 18 Synthesized peptide PDSPLNCDSGNLLRSGDIRGY
Gene name Locus number Annotation Mol. wt (kDa) Antigen Fragment length (position)/peptide sequence
HSP Os09g30418.1 Heat shock protein 94 Expressed protein 182 amino acids (8–189)
UBQ Os06g46770.2 Polyubiquitin containing 7 ubiquitin monomers 60 Expressed protein 150 amino acids (1–150)
TUB Os01g59150.1 Tubulin beta-4 chain 50 Synthesized peptide QYQDATADEEGEYEDEEQQ
eEF-1α Os03g08020.1 Elongation factor 1-α 49 Expressed protein 148AA(301–448)
eIF-4α Os02g05330.1 Eukaryotic initiation factor 4-α 47 Synthesized peptide DAKHYDSKMQELLHQGDNEE
SAMS Os05g04510.1 S-Adenosylmethionine synthetase 1 43 Expressed protein 150 amino acids (151–300)
ACT Os03g50890.1 Actin-1 42 Expressed protein 128 amino acids(251–378)
GAPDH Os04g40950.1 Glyceraldehyde-3-phosphate dehydrogenase, cytosolic 37 Synthesized peptide DLVSTDFQGDNRSSIFDAKAGI
UBC Os02g42314.2 Ubiquitin-conjugating enzyme E2 18 Synthesized peptide PDSPLNCDSGNLLRSGDIRGY

Rice reference protein candidates

Gene name Locus number Annotation Mol. wt (kDa) Antigen Fragment length (position)/peptide sequence
HSP Os09g30418.1 Heat shock protein 94 Expressed protein 182 amino acids (8–189)
UBQ Os06g46770.2 Polyubiquitin containing 7 ubiquitin monomers 60 Expressed protein 150 amino acids (1–150)
TUB Os01g59150.1 Tubulin beta-4 chain 50 Synthesized peptide QYQDATADEEGEYEDEEQQ
eEF-1α Os03g08020.1 Elongation factor 1-α 49 Expressed protein 148AA(301–448)
eIF-4α Os02g05330.1 Eukaryotic initiation factor 4-α 47 Synthesized peptide DAKHYDSKMQELLHQGDNEE
SAMS Os05g04510.1 S-Adenosylmethionine synthetase 1 43 Expressed protein 150 amino acids (151–300)
ACT Os03g50890.1 Actin-1 42 Expressed protein 128 amino acids(251–378)
GAPDH Os04g40950.1 Glyceraldehyde-3-phosphate dehydrogenase, cytosolic 37 Synthesized peptide DLVSTDFQGDNRSSIFDAKAGI
UBC Os02g42314.2 Ubiquitin-conjugating enzyme E2 18 Synthesized peptide PDSPLNCDSGNLLRSGDIRGY
Gene name Locus number Annotation Mol. wt (kDa) Antigen Fragment length (position)/peptide sequence
HSP Os09g30418.1 Heat shock protein 94 Expressed protein 182 amino acids (8–189)
UBQ Os06g46770.2 Polyubiquitin containing 7 ubiquitin monomers 60 Expressed protein 150 amino acids (1–150)
TUB Os01g59150.1 Tubulin beta-4 chain 50 Synthesized peptide QYQDATADEEGEYEDEEQQ
eEF-1α Os03g08020.1 Elongation factor 1-α 49 Expressed protein 148AA(301–448)
eIF-4α Os02g05330.1 Eukaryotic initiation factor 4-α 47 Synthesized peptide DAKHYDSKMQELLHQGDNEE
SAMS Os05g04510.1 S-Adenosylmethionine synthetase 1 43 Expressed protein 150 amino acids (151–300)
ACT Os03g50890.1 Actin-1 42 Expressed protein 128 amino acids(251–378)
GAPDH Os04g40950.1 Glyceraldehyde-3-phosphate dehydrogenase, cytosolic 37 Synthesized peptide DLVSTDFQGDNRSSIFDAKAGI
UBC Os02g42314.2 Ubiquitin-conjugating enzyme E2 18 Synthesized peptide PDSPLNCDSGNLLRSGDIRGY

Gene cloning, protein expression, and protein purification

PCR was carried out using plasmids from the rice cDNA libraries as templates. The indicated restriction enzymes were used to digest the amplicons and vectors, which were then gel purified. Next, the ligated products were transformed into E. coli DH5α, and the recombinants were verified using sequence analysis (Beijing Genomics Institute, Beijing, China). The recombinants were transformed into the E. coli expression strain ER2566 or BL21, and cultured overnight in LB medium supplemented with kanamycin (50 μg ml −1 ) at 37 °C. Cultures were diluted 1:100 with fresh Luria–Bertani medium (LB medium) supplemented with kanamycin (50 μg ml −1 ) and 1% glucose, and cultured at 37 °C to OD600 0.6–0.8. Next, isopropyl-β-d-thiogalactopyranoside (IPTG 0.4 μM) was added for 3 h to induce the expression of fusion proteins. The bacterial cells were harvested, ruptured by using sonication, and purified by nickel column chromatography. The target proteins were then separated by using SDS–PAGE and stained with Coomassie blue.

Antibody generation

The polyclonal antibodies were generated by immunizing healthy rabbits using the purified fusion proteins or the synthesized peptides as antigens. The protein conjugations, immunizations, and antiserum purifications were carried out by BPI (Beijing Protein Innovation Co., Ltd, Beijing, China).

Extraction of rice proteins and determination of their concentration

Rice tissue was ground into a fine powder in liquid nitrogen. An 800 μl aliquot of extraction buffer [62.5 mM TRIS-HCl (pH 7.4), 10% glycerol, 0.1% SDS, 2 mM EDTA, 1 mM phenylmethylsulphonyl fluoride (PMSF), 5% (v/v) β-mercaptoethanol] was added to each 300mg powder sample. The mixture was vortexed and then chilled on ice for 10 min. Samples were centrifuged at 12 000 rpm for 10min at 4 °C, and the supernatant was collected and stored at –70 °C. The protein concentrations of the rice samples were determined using the Bradford method ( Bradford, 1976). An equal amount of rice protein was loaded and separated by SDS–PAGE and then stained by Coomassie blue.

Western blotting and signal quantification analysis

Equal amounts of rice protein from different tissues/organs were separated using SDS–PAGE and electrotransferred to a PVDF membrane (Millipore Corporation, Bedford, MA, USA) at 100 V for 60 min. The membrane was immersed in 5% non-fat milk in a TTBS solution [0.2 M TRIS-HCl (pH 7.6), 1.37 M NaCl, 0.1% Tween-20] for 1h at room temperature. The proteins were incubated with the polyclonal antibodies in 5% non-fat milk in a TTBS solution for 3 h at room temperature and subjected to three 5 min rinses in a TTBS solution. The membrane was then incubated with a horseradish peroxidase-conjugated goat anti-rabbit antibody (Zhongshan Goldenbridge Biotechnology Co., Ltd, Beijing, China) for 1 h at room temperature, and subjected to three 5 min rinses in a TTBS solution. The blot was developed with a SuperECL Plus kit (Applygen, Beijing, China), and the signal was exposed with X-ray film.

The images were scanned and the intensity of each band was captured using an ImageMaster 2D Platinum version 5.0 (GE Healthcare Amersham Bioscience). The intensity of each band was standardized as a percentage of the total intensity and the results were referred to as a relative volume that represents the relative expression abundance of the gene in the samples tested. The relative expression abundance was used to evaluate protein expression stability. Western blotting and quantification analysis were performed in at least three biological replications.

Analysis of protein expression stability

The protein expression stability was evaluated by geNorm v.3.5 (http://medgen.ugent.be/∼jvdesomp/genorm/) ( Vandesompele et al., 2002) and Microcal Origin 6.0 software (Microcal Software, Northampton, MA, USA). The relative protein expression values were imported into geNorm, which calculates the gene expression stability [M value or the mean of the standard variation of a given candidate gene relative to all other genes in the given set of samples ( Murthi et al., 2008 Silveira et al., 2009)] to rank proteins in various tissue samples. The lower the M value, the more stable the protein expression. In addition, the relative values of protein expression were imported into Microcal Origin 6.0, which generates a box plot to estimate the expression stability of potential reference proteins.

Generation of western blotting standard curves and determination of the concentration of reference proteins in rice

The concentration of recombinant proteins was plotted against the western blotting signal generated by the corresponding antibodies in order to estimate the linear range of detection and the lower limits of detection of the recombinant proteins. Then, a series of diluted recombinant proteins and total rice proteins were assayed in the same western blotting membrane to generate the standard curve. The standard curve was used to calculate the protein concentration and percentage of reference proteins in rice. A series of dilutions of total rice proteins were also analysed using western blotting in order to determine the lower limits of detection for the rice reference proteins.

Transcriptional analysis of rice reference genes

Transcriptional data on rice leaf, root, stem, panicle, and seed were downloaded from the MPSS (http://mpss.udel.edu/rice/) ( Nakano et al., 2006) and EST databases (http://www.ncbi.nlm.nih.gov/projects/dbEST/) ( Boguski et al., 1993). The transcriptional level was divided into four grades based on the intensity of the expression signal in order to compare it with the western blotting results.


The Basis of Western Blot

There are many “Blot” techniques in molecular biology such as southern blot, northern blot, eastern blot and western blot. The principle and process of these blot techniques are similar. Southern and northern blot are nucleic acid blotting techniques, of which southern blot is for detecting a specific DNA sequence in DNA samples and northern blot is to study gene expression by detection of RNA (or isolated mRNA) in a sample. Eastern blot is used to analyze protein post translational modifications (PTM) such as lipids, phosphomoieties and glycoconjugates.

Different from those blot technique described above, western blot, also called protein immunoblot, is an analytical technique used to detect specific proteins in a given sample such as cell lysates, tissue homogenate or extract. It is widely used in molecular biology. The bases of western blot identification are two distinguishing properties: molecular weight and antibody binding specificity. It uses gel electrophoresis (usually SDS-PAGE) to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide. And then transferred the protein sample to a membrane (typically nitrocellulose or PVDF), where they are probed with labeled-antibodies and stained with dyes for visualization and directly identified by N-terminal sequencing, mass spectrometry or immunodetection. Immunodetection involves the identification of a protein through its reaction with a specific antibody. Through spatial resolution, this method provides molecular weight information on individual proteins and distinguishes isoforms, alternate processing products, and other post-translationally modified forms.

2. Basic principle of western blot

The basic principle of western blot are protein electrophoresis and ELISA. Electrophoresis is a commonly used method for separating proteins on the basis of size, shape or charge. Proteins are large molecules with charge, they can migrate in the polyacrylamide gels under electric field. The migrating speed depends on the molecular weight of proteins: heavy proteins move slower than light proteins. This is just like the runners running through a jungle, the polyacrylamide molecule in the gel is like the branches along the sideline, which could slow the runner down. The bulky runner would be affected more by the “branches” than the small runner so that they runs slower. That’s why we could separate those runner proteins. If we load the mixed protein samples on one side of the gel, add a constant electric field on the gel, and let the protein migrating on the gel for a period of time, the mixed protein sample could be separated into different bands on gel (Figure 1). If we put several proteins with known molecular weight under the same electrophoresis condition along with the tested samples as control, we may further measure the molecular weight of proteins within the tested samples by compare the location of sample proteins with control proteins. These control proteins are commonly known as molecular weight marker or protein ladders.

Figure 1. Protein electrophoresis: proteins separated under electric fields based on different molecular weight.

When the proteins were successfully separated, we still use an electric field to transfer the protein bands onto another polyvinylidene fluoride (PVDF) or nitrocellulose (NC) membrane for the following detection. After the proteins band transferred onto PVDF/NC membrane, we use anti specific protein primary antibodies to probe the sample protein, and then use a labeled secondary antibody for further visualization (Figure 2). This procedure is basically an indirect ELISA process (know more about indirect ELISA at ELISA Guide).

Creative Diagnostics provides a variety of high quality primary antibodies and secondary antibodies for western blot use.

Figure 2. Indirect ELISA procedure. To probe and visualize the separated protein bands.

3. The experimental equipment of western blot

As western blot is a very mature technology in molecular biology, there are a full set of commercialized reagent and equipment that support the experiment of WB (Figure 3). Here we list several frequently used tools and materials for western blot:

  1. Gel: usually made of polyacrylamide, need precast before western blot.
  2. Gel comb: used for toothing one side of the gel as sample loading area.
  3. Gel cassette: used for gel formation.
  4. Gel knife: help to move the gel from cassette.
  5. Electrode chamber: used for holding the gel cassette and cathode buffer.
  6. Electrophoresis tank: used for holding the electrode chamber and anode buffer
  7. Power source: provide a constant electric field
  8. Cassette: clamp the multilayer films when doing protein transfer
  9. Sponges: protect the gel and membrane, keep a wet and conductive condition.
  10. Filter paper: protect the gel and membrane, keep a wet and conductive condition.
  11. PVDF/NC membrane: receive the proteins.
  12. Rolling brush: exclude bubbles within the multilayer membrane.
  13. Washing dish: hold the membrane for washing.

Other general equipment such as shaker/incubator for antibody incubation and imager for display the gel result are not listed here but also important for western blot experiment.

Figure 3. Western blot equipments.

Western blot is often used in research to separate and identify proteins. In this technique a mixture of proteins is separated based on molecular weight through gel electrophoresis. These results are then transferred to a membrane producing a band for each protein. The membrane is then incubated with labeled antibodies specific to the protein of interest. The unbound antibody is washed off leaving only the bound antibody to the protein of interest. The bound antibodies are then detected by developing the film. As the antibodies only bind to the protein of interest, only one band should be visible. The thickness of the band corresponds to the amount of protein present thus doing a standard can indicate the amount of protein present. The general process of western blot includes at least 9 steps as following:

Detailed information of each steps are discussed in Western blot protocols.

Although western blot is relatively an old, mature technology, there are still researchers working on it to upgrade this technique. Hughes et al. from University of California (UC) Berkeley, successfully couple western blotting with single-cell analysis, which enables the simultaneous analysis of

2,000 individual cells. Single-cell protein analysis techniques lack resolution, sensitivity or specificity, or require protein tagging. Western blotting avoids these pitfalls but is not amenable to single-cell analysis. This progress enable us use western blot to investigate single cell proteins.

Their array-based technique uses a slide coated with polyacrylamide gel and patterned with thousands of micro wells, into which a cell suspension is seeded by gravity-driven cell settling, resulting in single-cell occupancy in 40–50% of the wells. Intracellular proteins are then solubilized in the wells, subjected to thin-gel electrophoresis and immobilized. Subsequently, the serial stripping and re-probing of antibodies enables multiplexed analyses of proteins.


Antibody Validation

Antibodies are one of the important reagents in life sciences and clinical medicine. Although antibodies have been widely used, there is a lack of established criteria to guide how they should be validated before use. As a result, many commercial antibodies have not been fully validated prior to use and/or lack of further confirmation resulting in failed or unreplicable results, even projects being abandoned, resulting in significant time, money and sample loss. Therefore, the establishment of antibody verification standards has become an urgent need.

What Is Antibody Validation?

Antibody validation consists primarily of the following components: demonstrating specificity (the ability of antibodies to distinguish between different antigens), proving affinity (intensity of antibody binding epitopes), and finally demonstrating reproducibility. However, although this definition of antibody validation is reasonable, there are still some problems with the widespread application or implementation of validation standards.

The task of verifying the antibody should have been done by the antibody supplier. However, although the seller is responsible for the quality of the reagents sold, antibody performance may be affected by a number of other factors. For example, antibodies may change during transport due to improper storage at low temperatures, or although they can be successfully validated in a general biologically relevant system, end users need to use it in their own experimental systems. Therefore, the end user also needs to perform secondary verification of the antibody.

By taking a few small steps, the researcher can effectively reduce the risk of the experiment in subsequent experiments. First, the researcher should be aware of the usage information provided in the product specification, including the application that has been validated for it, the appropriate protocol and the recommended dilution.

How Can Antibodies Be Validated?

There are also different methods for antibody validation for specific applications, including Western blot (WB), immunohistochemistry (IHC), immunocytochemistry (ICC), immunofluorescence (IF), ELISA, immunoprecipitation (IP), chromatin immunoprecipitation. (ChIP) and peptide arrays, etc. In terms of verification, each assay has its advantages and disadvantages (Table 1).

Table 1. Advantages and disadvantages of several antibody validation methods.

Assay Advantage Disadvantage
Western Blot Easy and simple assay
Ideal for denatured proteins
Difficult to optimize
Time-consuming
A small number of antibodies can be tested per run
ELISA Quantitative assay
High throughput
Does not determine if the antibody is specific or cross-react
IHC/ICC Routinely available and relatively inexpensive Difficult standardization
Epitope accessibility can vary in fixed tissues
Difficult to quantitate
IF High throughput
Easy to optimize
Does not determine if the antibody is specific or cross-reacts
siRNA knockdown KD cell lines can be used in all assays (WB, ICC/IHC, Flow cytometry) KD is transient
Difficult to optimize-requires several siRNA sequences
Knockout cell lines and mouse models Best negative controls, since they guarantee no expression of the target gene
May be used in all assays (WB, ICC/IHC, Flow cytometry)
Cell lines for specific genes are not always available or lethal
KO mouse models take over a year to develop and are often non-viable
MS Confirms specificity
High throughput
Requires use of mass spectrometer and trained personnel
SPR Real-time analysis
Label-free
Highly sensitive
Requires immobilization
Requires meticulous experimental design
High sample consumption
MST Rapid assay (KD in 10 min)
Low sample consumption (pM/nM) and small volume (<4 ul)
Immobilization free-in solution measurements
Label-free (optional)
Requires specialized equipment

Which method should be used and how several methods are used to validate antibodies depends not only on sensitivity, specificity and reproducibility, but also on the environment in which the particular antibody is to be used. This means that if a given antibody is only intended for WB, they only need to be tested in this case. However, if you need to use it in other assays, you should also test it again in the application environment. This helps save time and cost associated with the antibody validation process.

Screening for Antibody Specificity

Specificity is a measure of the ability of an antibody to distinguish between different antigens. Even if the antibody may be sensitive to the target protein, it will show a lack of specificity due to cross-reactivity with other proteins. In this sense, it is a basic requirement for a good antibody to confirm that the antibody specifically binds to the target protein.

Several questions need to be identified before analyzing the specificity of a given antibody. The first is what are the types of immunogens that are used to produce this antibody? How is the target protein in the sample to be analyzed constructed? The first question is not easy to answer, especially if the antibody manufacturer does not disclose the type of immunogen. The reason for understanding this information is that the specificity of an antibody depends in part on whether the type of immunogen is a synthetic peptide or a purified protein. Since synthetic peptides do not necessarily summarize the 3-D structure or post-translational modification of native proteins, antibodies produced using synthetic peptides may not recognize native proteins. If the immunogen is a purified protein, the denatured protein is difficult to bind to the antibody. Thus, when the immunogen is a synthetic peptide, the antibody can be applied to WB but not to IP or IHC.

The Preferred Method for Antibody Specific Validation - WB

WB is considered to be the first step in the evaluation of new antibodies because it is the ideal assay for specific validation of denatured protein antibodies. However, in experimental assays where the antigen is in its natural conformation (eg, IHC), it is not the ideal standard method for antibody binding. Because antibodies may recognize a particular epitope in fresh tissue, but different epitopes are recognized in the fixed tissue, the method used to fixed tissue complicates the problem. This happens because unexposed epitopes in natural proteins become accessible after fixed processing and vice versa. If you only need to verify the recognition of the denatured antigen by the antibody or whether it is suitable for WB, then the assay can be used as a first step of verification. The result when the antibody is specific for the selected target is the observation of a band on a target of known molecular weight. If multiple bands are present, they may represent different post-translational modification states, decomposition products or splice variants, or may also be non-specific binding. Therefore, the results of these experiments may appear to be incapable of determining antibody specificity.

Another disadvantage of WB is its low detection throughput because it usually only tests one antibody at a time. However, recently, by converting WB to a capture format, a high-throughput detection method called PAGE-MAP was developed for antibody verification. In this method, a biotinylated protein sample is subjected to PAGE, and then the size-separated protein is incubated with a microsphere-based barcode antibody array for multiplex IP, and the captured protein is labeled with streptavidin for flow through Magnetic cytometry (ie, microsphere affinity) was performed for magnetic bead detection. In addition, size-separated proteins can also be used for parallel readout shotgun mass spectrometry (MS), which can be used to roughly estimate specificity and for selection of antibody target levels sufficient for immunoprecipitation. This new method is powerful, screens thousands of antibodies, recognizes antibodies that bind to the same protein, and provides a means for large-scale antibody validation.

Figure 1. Overview of the western blotting procedure.

In addition to the WB method, the use of blocking peptides to assess antibody specificity is also a common approach, particularly for IHC. These peptides are identical to the peptides used to produce antibodies, and when over-incubated with them, they can be used as immunoneutralizing antibodies. In the validation experiment, the unneutralized antibody was used as a control to stain the sample tissue. If the antibody is specific, incubation with a blocking peptide will result in the disappearance of staining on the tissue. However, this assay has the disadvantage that it does not verify the selectivity of the antibody for the antigen, since the non-specific binding activity of the antibody will also be inhibited by the blocking peptide. Thus, blocking peptides can prove that antibodies are bad, but they do not prove that antibodies must be good.

In the process of antibody validation, the key to demonstrating antibody specificity is the correct use of controls. As in the antibody verification experiments on the cannabinoid CB2 receptor, it has been found that the conventional practice of using only a positive control to verify an antibody is insufficient to ensure the reliability of the antibody. In this validation study, although many techniques (eg, WB, mass spectrometry and blocking peptides) and excellent positive controls were used to obtain a number of positive results indicating the effectiveness of anti-CB2 antibodies. However, when knocking out a negative control to test antibodies, this antibody was found to be non-specific for the CB2 receptor protein. A similar situation may occur due to the similarity of relative epitope regions between proteins, or because antibodies have multiple epitopes. The best negative control is knockout cells/animals that do not express the protein of interest, and closely related proteins can also serve as good negative controls. The best positive control is a cell which non-expression the target protein and transfected with the protein of interest. If knocking out the cells is too difficult to obtain, then siRNA or shRNA knockout cells can be selected as controls. Although negative control is necessary, there is also a need for positive control.

Determination of Antibody Affinity

Antibody binding affinity is another parameter that can be used for antibody validation. It refers to the strength of the binding of an antibody molecule to an epitope. It is usually reported as the equilibrium dissociation constant (KD), which is the ratio of the antibody dissociation rate or koff (the rate of dissociation from the antigen) to the antibody association rate or antibody binding rate Kon(the rate of binding to the antibody).

Affinity determination of monoclonal antibodies can be performed with high precision because they are selective only for the same epitope, but in the case of polyclonal antibodies, since the epitopes they detect are heterogeneous and are various A mixture of antibodies with different affinities. Therefore, only the average affinity can be obtained. In order to determine antibody affinity, the following methods have existed. These include ELISA-based methods, as well as other biophysical methods such as microscale thermophoresis (MST) and surface plasmon resonance (SPR).

ELISA is the most popular method for studying antibody affinity. They do not require the use of large amounts of antibodies and antigens, nor do they require purification of the protein. In this method, a fixed concentration of antibody is incubated with the antigen in solution until a steady state is reached. Then, the concentration of the unbound antibody was measured by an indirect ELISA. This method requires prior preliminary experiments to determine the concentration of antibody used within the linear range of the ELISA reaction, and only a small fraction of the total free antibody in the solution remains on the plate (so that the measurement does not significantly affect the equilibrium in the solution).

MST is a biophysical method that detects the affinity of antibody molecules over a wide range of concentrations. It judges the affinity between molecules by measuring the movement of molecules along the infrared laser-induced microscopic temperature gradient. This movement depends on many factors, including the hydrated shell, charge and molecular size. These molecules are initially uniformly distributed in solution and free to diffuse. When the infrared laser is turned on, the unbound molecules usually move out of the heating point. The binding of one molecule to another (such as antibodies and antigens) causes the movement of the entire temperature gradient to change. The movement of the molecule can then be followed by fluorescent labeling to derive affinity parameters for the antibody molecule.

Figure 2. Determination of antibody affinity by Microscale Thermophoresis (MST).

SPR is an optical technique for detecting molecular interactions in which one molecule is immobilized in a metal film and the other molecule is mobile. The combination of these molecules changes the refractive index of the film. Therefore, when polarized light strikes the film, the extinction angle of the light changes and can be monitored by an optical detector (Figure 3).

Figure 3. Determination of antibody affinity by Surface Plasmon Resonance (SPR).

Antibody Reproducibility

Finally, verify the reproducibility of the antibody, which is an essential part of antibody detection. A common misconception in antibody testing is to assume that antibodies produce similar results, whether from the same or different batches or from different manufacturers. One of the most shocking examples is David Rim of Yale University. He developed an antibody-based assay to guide the effective treatment of melanoma and is ready to apply the assay to the clinic. However, when he ordered a new set of antibodies from the same company, he could not reproduce the original results and had to give up his work. That is to say, using antibodies from the same supplier over time, even if only the batch is different, will produce different results. Therefore, it is critical for researchers to always test for antibody repeatability. This becomes especially important when using polyclonal antibodies, as products from the same item number from the supplier may mean different antibodies.

In summary, in order to ensure that the antibody meets the specificity and affinity and reproducibility required for its use, it is necessary to perform a secondary verification strictly according to the antibody detection standard. Only in this way can the reliability of the experimental data be guaranteed.