Case Study 1: eXceptional Specificity of XP® Monoclonal Antibodies
Phospho-Met (Tyr1234/1235) (D26) XP® Rabbit mAb #3077 Compared to a Competitor’s Product
A common problem with many commercially available antibodies directed against phosphorylated receptor tyrosine kinases (RTKs) is crossreactivity with related active RTKs, resulting in a false positive signal. Comparison of Phospho-Met (Tyr1234/1235) (D26) XP® Rabbit mAb #3077 from Cell Signaling Technology (CST) with a competitor’s product illustrates the value of CST’s high performance XMT technology-derived products combined with CST’s extensive product validation.
The following validation steps by CST scientists demonstrate superior specificity of #3077.
- Side by side comparison of CST #3077 and a competitor’s product on HCC827 xenograft gives the appearance of specific staining for both products (Figure 1).
- Western analysis confirms a single and specific band of the appropriate molecular weight for #3077, but not for the competitor’s antibody (Figure 2).
- Paraffin-embedded cell pellets treated with various RTK inhibitors and stimulators confirm specificity by IHC of #3077 but not of the competitor’s antibody (Figure 3).
- Specificity, sensitivity and performance of #3077 was further confirmed by IHC using tissue samples and cell pellets (Figure 4 and 5).
- The possibility of cross-reactivity with the activated form of Ron, another member of the Met kinase family, was addressed by western analysis and IHC (Figure 6).
In conclusion, CST’s Phospho-Met (Tyr1234/1235) (D26) XP® Rabbit mAb #3077 shows the exceptional specificity necessary for superior performance in applications such as IHC. This conclusion can only be drawn through the extensive product validation with the scientifically appropriate controls as performed at CST.
Figure 3. Immunohistochemical analysis of paraffin-embedded MKN45 cells treated with the Met inhibitor SU11274 showed no staining as compared to the control using both Phospho-Met (Tyr1234/1235) (D26) XP® Rabbit mAb #3077 and the competitor’s antibody. EGF treatment and HRG treatment of MDA-MB-468 and T47D breast carcinoma cell lines, respectively, showed no increase in staining compared to control using #3077. In contrast, the competitor’s antibody detected non-specific staining with both treatments, indicating cross-reactivity with other RTKs.
Figure 1. Immunohistochemical analysis of paraffin-embedded HCC827 xenograft comparing #3077 (left) and a competitor’s product (right) gives the appearance of specific staining for both products.
Figure 2. A single band at 145 kDa was observed by western blotting in HGF-stimulated, but not in unstimulated A431 cells using #3077. Extracts treated with growth factors that activate other RTKs or that overexpress other RTKs or cytoplasmic tyrosine kinases were negative (top). By comparison, the competitor phospho-Met antibody recognizes several nonspecific bands (bottom). Both membranes were developed on the same film with the same exposure time (10 seconds).
Figure 4. Immunohistochemical analysis of src-transfected NIH/3T3 cells using Phospho-Src Family (Tyr416) Antibody (left) or #3077 (right). No staining was observed with #3077 indicating that the antibody does not cross-react with Src phosphorylated at Tyr416 by IHC.
Figure 5. Phosphatase treatment of paraffin-embedded human lung carcinoma confirms phospho-specificity of #3077.
Figure 6A. The purified active Ron kinase can be detected with various phospho-Ron antibodies by western blotting and phospho-tyrosine antibody, but not #3077, indicating no cross-reactivity of #3077 with Ron by western analysis. Exposure time: 60 seconds.
Figure 6B. Immunohistochemical analysis of xenografts from 3T3-Met (left) and 3T3-Ron cells (right) using #3077 demonstrates that #3077 does not cross-react with activated Ron by IHC. Image courtesy of Pfizer, Inc.
