Western blotting provides a crucial screen of antibody specificity that should be performed before using the antibody in IHC or ICC.
Contributed by Mike BrowningMisconceptions exist in the research community about the relevance of Western blot data in IHC and ICC.
As we have discussed previously in this blog, there is widespread concern about antibody validation. Of course some forms of validation using multiple antibodies or knockouts are not available for the majority of targets of interest to researchers. But western blots are an excellent form of validation that can be readily performed by almost any lab. Thus it is puzzling why so many immunostaining papers fail to use this straightforward antibody validation tool. I would suggest that the failure to use western blots (WBs) as validation tool before immunostaining most likely results from a widespread misunderstanding in the research community about the relevance of WB data to immunostaining. Some have suggested that the detection environment in WB with the denaturing effects of SDS is so different from that in immunostaining as to make WB data irrelevant to immunostaining. However, as emphasized by Forsström et al. (1) it is important to understand that both WB and IHC use denaturing conditions.
...any antibody that yields multiple bands in Western blots raises a critical red flag, and the antibody should most likely not be used in IHC or ICC...
While exceptions exist (2-4), a large body of evidence points to a high correlation between positive WB data for an antibody and good data for the same antibody in IHC, ICC and IF. (5-8). As argued by Kurien et al (8) “immunoblotting is a must to determine specificity of antibodies used for Immunohistochemistry (IHC).” The Journal of Endocrinology and the Journal of Histochemistry and Cytochemistry both editorialize in favor of using WB as an initial antibody screen (9;10). Both argue that any antibody that yields multiple bands in WBs raises a critical red flag and that the antibody should not be used in IHC unless some other test can be used to validate the antibody.
Antibodies that give a single band in Western blot almost always give good signal in immunostaining.
After making over 500 antibodies over the past few decades, we have found that more than 90% of the antibodies that gave a single band signal in WB also gave a good signal immunostaining. See for example Fig.1 where WB and IHC staining of an antibody to synapsin I, a neuron specific synaptic vesicle associated protein, is shown. As shown clearly in the figure, the synapsin antibody specifically labels only the synapsin I doublet in the WB. Similarly the IF image shows the same synapsin antibody exhibiting specific punctuate labeling characteristic of the localization of the synaptic vesicle associated protein.
Western blotting, while not perfect, is a very good antibody validation tool.
Antibody validation by WB is certainly not perfect. However, it is important to realize that WBs provide very important validation tool particularly given the fact that no other validation method is available for most targets. The ideal antibody validation tool is of course the use of knock-out animals in the immunological methods of interest (however see (11;12) for some limitations on the use of knockouts in antibody validation). Thus when knockouts are available they should almost always be used in preference over WB. Unfortunately, knockouts are available for only a very small percentage of the protein targets of interest. Consequently, it seems illogical to let the fact that WB validation is not a perfect validation tool to limit its use as a very good antibody validation tool. This is particularly true at a time when the results of many antibody studies being published use antibodies with little or no validation, leading to data that is flawed and cannot be reproduced.
If Western blots are going to be used as a validation tool it is essential that best practices be utilized in the WB assay.
We have described our basic WB protocol in our support section. However two other issues are of critical importance. First it is essential that a true tissue or cellular lysate be used rather than recombinant proteins for antibody validation in WB. This issue is discussed in detail in the blog on recombinant proteins. Lastly it is also critical that the lysate buffer completely solubilize all the proteins in the sample. We strongly recommend the use of 1% SDS in the lysis buffer as discussed in the blog on Lysis Buffers.
Reference List
- Forsstrom B, Axnas BB, Rockberg J, Danielsson H, Bohlin A, Uhlen M. Dissecting antibodies with regards to linear and conformational epitopes. PLoS.One. 2015;10:e0121673.
- Herrera M, Sparks MA, Alfonso-Pecchio AR, Harrison-Bernard LM, Coffman TM. Lack of specificity of commercial antibodies leads to misidentification of angiotensin type 1 receptor protein. Hypertension 2013;61:253-8.
- Yu W, Hill WG. Lack of specificity shown by P2Y6 receptor antibodies. Naunyn Schmiedebergs Arch.Pharmacol. 2013;386:885-91.
- Baek JH, Darlington CL, Smith PF, Ashton JC. Antibody testing for brain immunohistochemistry: brain immunolabeling for the cannabinoid CB(2) receptor. J.Neurosci.Methods 2013;216:87-95.
- Schuster C, Malinowsky K, Liebmann S et al. Antibody validation by combining immunohistochemistry and protein extraction from formalin-fixed paraffin-embedded tissues. Histopathology 2012;60:E37-E50.
- Egelhofer TA, Minoda A, Klugman S et al. An assessment of histone-modification antibody quality. Nat.Struct.Mol.Biol. 2011;18:91-3.
- Sawicka M, Pawlikowski J, Wilson S et al. The specificity and patterns of staining in human cells and tissues of p16INK4a antibodies demonstrate variant antigen binding. PLoS.One. 2013;8:e53313.
- Kurien BT, Dorri Y, Dillon S, Dsouza A, Scofield RH. An overview of Western blotting for determining antibody specificities for immunohistochemistry. Methods Mol.Biol. 2011;717:55-67.
- Saper CB. A guide to the perplexed on the specificity of antibodies. J.Histochem.Cytochem. 2009;57:1-5.
- Gore AC. Editorial: antibody validation requirements for articles published in endocrinology. E 2013;154:579-80.
- Lorincz A, Nusser Z. Specificity of immunoreactions: the importance of testing specificity in each method. J.Neurosci. 2008;28:9083-6.
- Watanabe M, Fukaya M, Sakimura K, Manabe T, Mishina M, Inoue Y. Selective scarcity of NMDA receptor channel subunits in the stratum lucidum (mossy fibre-recipient layer) of the mouse hippocampal CA3 subfield. Eur.J.Neurosci. 1998;10:478-87.
Further Reading
SDS-PAGE Demystified – The Science Behind All Those Bubbles!
Are Monoclonal Antibodies Really More Specific?