Proteins can be expressed in varying levels in different cell or tissue types. Relative expression testing methods exploit naturally occurring variable expression patterns in cell or tissue types to confirm specificity of the antibody to the target protein. Detection of the expected differential basal expression pattern of the target across tissue/cell models demonstrates antibody specificity. Relative expression of these proteins in different cell models can be analyzed using a variety of applications (immunofluorescence, western blotting, flow cytometry, etc).
The Advanced Verification badge is applied to products that have passed application and specificity testing. Advanced Verification testing of Invitrogen antibodies continues to expand across the portfolio. An antibody lacking the badge should be seen as an antibody that has not yet undergone testing – not a reflection of the specificity of the product. This badge can be found in the search results and at the top of the product specific webpages. Data supporting the Advanced Verification badges can be found in product specific data galleries.
Relative expression validation data
Antibody specificity was demonstrated by detection of differential basal expression of the target across cell models owing to their inherent genetic constitution. Expression of OCT4 was observed specifically in NTERA-2, NCCIT and F9, cell lines of embryonic origin, but not in the somatic tumor cell lines HEK-293, HeLa and A431, using Anti-OCT4 Recombinant Rabbit Monoclonal Antibody (Cat. No. 701756) in Western blot. OCT4 is involved in the maintenance of embryonic stem cell pluripotency, and its expression is lost at the blastocyst stage.
Figure 1. Western blot analysis was performed on whole cell extracts (30 µg lysate) of NTERA-2 (Lane 1), NCCIT (Lane 2), F9 (Lane 3), HEK-293 (Lane 4), A431 (Lane 5) and HeLa (Lane 6). The blots were probed with Anti-OCT4 Recombinant Rabbit Monoclonal Antibody (Cat. No. 701756, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Cat. No. A27036, 0.4 µg/mL, 1:2500 dilution).
Antibody specificity was demonstrated by detection of differential basal expression of the target across cell lines owing to their inherent genetic constitution. Expression of Her2 was observed in SK-BR-3 compared to MDAMB-231 using Her2 Monoclonal Antibody (Cat. No. MA5-13032) in ICC.
Figure 2. Immunofluorescence analysis of Her2 was performed using 70% confluent log phase SK-BR-3 cells (panels a to e) and MDA-MB-231 cells (panel f). The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Her2 Mouse monoclonal Antibody (Cat. No. MA5-13032) at 5 µg/mL in 0.1% BSA and incubated overnight at 4 degree Celsius and then labeled with Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Cat. No. A28175) at a dilution of 1:2000 for 45 minutes at room temperature.
Flow cytometry intrinsically facilitates the analysis of relative expression patterns in heterogeneous cell populations. Using gating techniques, verification of antibody binding can be determined by analyzing expression in unique cell types. In the example below (Figure 3), flow cytometry was used to look at staining of known markers representative of specific cell populations within whole blood. As expected based on known expression patterns, the CD3 antibody only stains a subset of lymphocytes (T cells), the HLA-DR antibody stains monocytes and a subset of lymphocytes (B cells), and the CD16 antibody stains all granulocytes, a subset of monocytes and a subset of lymphocytes (NK cells).
Figure 3. Normal human whole blood was surface stained with CD3 (clone UCHT1, left plot), HLA-DR (clone L243, middle plot), and CD16 (clone CB16, right plot). After staining, red blood cells were lysed using 1-step Fix/Lyse Buffer. Cells in the lymphocyte (purple histogram), monocyte (orange histogram), or granulocyte (blue histogram) gates were used for analysis.
In this second example (Figure 4), as expected based on known expression patterns, the Foxp3 antibody only stains a subset of the CD4+ T cells and not the CD8+ T cells.
Figure 4. Balb/c splenocytes were surface stained with CD3 (clone 17A2), CD4 (clone GK1.5) and CD8 (clone 53-6.7), followed by intracellular staining with Foxp3 (clone FJK-16s) using the Foxp3/Transcription Factor Staining Buffer Set and protocol. Lymphocytes in the CD3+CD8+ (blue histogram) and CD3+CD4+ (purple histogram) gates were used for analysis.
Verifying target specificity of Invitrogen antibodies using relative expression
Invitrogen antibodies that have been verified against relative expression are indicated with a “verified specificity” symbol in search results and on relevant product pages. The data showing the verification will be provided on each product page.
For Research Use Only. Not for use in diagnostic procedures.