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 a 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. 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 Immunohistochemirty (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.
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
So in summary, 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. Having said that, if WBs are going to be used
as a validation tool it is essential that best practices be utilized in the WB
assay. In the next few days we will post
suggestions for these best practices and how to avoid pitfalls in using WB for
antibody validation.
Reference List
1. 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.
2. 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.
3. Yu W, Hill WG. Lack
of specificity shown by P2Y6 receptor antibodies. Naunyn Schmiedebergs
Arch.Pharmacol. 2013;386:885-91.
4. 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.
5. 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.
6. Egelhofer TA, Minoda
A, Klugman S et al. An assessment of histone-modification antibody quality.
Nat.Struct.Mol.Biol. 2011;18:91-3.
7. 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.
8. 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.
9. Saper CB. A guide to
the perplexed on the specificity of antibodies. J.Histochem.Cytochem.
2009;57:1-5.
10. Gore AC. Editorial:
antibody validation requirements for articles published in endocrinology. E
2013;154:579-80.
11. Lorincz A, Nusser Z.
Specificity of immunoreactions: the importance of testing specificity in each
method. J.Neurosci. 2008;28:9083-6.
12. 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.
