Antibody Validation by Western Blotting

Abstract

Validation of antibodies is an integral part of translational research, particularly for biomarker discovery. Assaying the specificity of the reagent (antibody) and confirming the identity of the protein biomarker is of critical importance prior to implementing any biomarker in clinical studies, and the lack of such quality control tests may result in unexpected and/or misleading results.Antibody validation is the procedure in which a single antibody is thoroughly assayed for sensitivity and specificity. Although a plethora of commercial antibodies exist, antibody specificity must be extensively demonstrated using diverse complex biological samples, rather than purified recombinant proteins, prior to use in clinical translational research. In the simplest iteration, antibody specificity is determined by the presence of a single band in a complex biological sample, at the expected molecular weight, on a Western blot.To date, numerous Western blotting procedures are available, based on either manual or automated systems and spanning the spectrum of single blots to multiplex blots. X-ray film is still employed in many research laboratories, but digital imaging has become a gold standard in immunoblotting. The basic principles of Western blotting are (a) separation of protein mixtures by gel electrophoresis, (b) transfer of the proteins to a blot, (c) probing the blot for a protein or proteins of interest, and (d) subsequent detection of the protein by chemiluminescent, fluorescent, or colorimetric methods. This chapter focuses on the chemiluminescent detection of proteins using a manual Western blotting system and a vacuum-enhanced detection system (SNAP i.d.™, Millipore).

Phosphoproteomic analysis reveals Smad protein family activation following Rift Valley fever virus infection

“…Antibodies were validated for their use on the array by Western Blot to determine antibody specificity. Only antibodies showing a single band at the expected molecular weight were used on the arrays [ 28 ]. Biotinylated anti-rabbit (Vector Laboratories, Inc.) or anti-mouse secondary antibody (CSA; Dako Cytomation) was used in conjunction with GenPoint ™ kit (Dako Cytomation), a commercially available tyramide-based signal amplification system.…”

Systematic quantification of synapses in primary neuronal culture

“…To gain more certainty about labeling performance, and provide a reference for the field, we have now explored a variety of benchmarking methods to evaluate the specificity of a set of antibodies raised against synaptic proteins. Although extensively used (42), we found that western blotting is not useful for routine antibody validation since neither the presence of additional bands (alternative splice forms) nor the absence of a specific band (epitope masking) can truly predict non-specificity. Therefore, we first introduced a segmentation-independent method to assess the staining performance of individual antibodies in immunofluorescence images.…”

<p>Exosomes Derived from Mesenchymal Stem Cells Protect the Myocardium Against Ischemia/Reperfusion Injury Through Inhibiting Pyroptosis</p>

“…As previously reported, 29 cell samples were cracked in RIPA lysis buffer plus PMSF on the ice, and total protein was measured with protein assay kit (BCA; Santa Cruz, California, USA). Equal amounts of total protein were separated in SDS-PAGE, and transferred into PVDF membranes (Millipore, USA) and incubated with prepared antibodies.…”