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Relative Expression Antibody Validation*
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. Certain proteins show changes in their expression levels or sub-cellular localization across different cell phases which can be used to verify the specificity of the antibody. In the case of antibodies against differentiation markers, the expression of the target following the differentiation into a specific cell type is an excellent method to show specificity. Relative expression of these proteins, due to target biology, can be analyzed using a variety of applications (i.e., immunofluorescence, western blotting, flow cytometry, etc.). This method of verification can be effectively used for validation of both primary and secondary antibodies as described in the examples below.
Relative expression validation data
Demonstration of primary antibody specific by relative expression across cell models
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 OCT4 Recombinant Rabbit Monoclonal Antibody (3H8L6) (Cat. No. 701756) by western blot analysis. 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) 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 OCT4 Recombinant Rabbit Monoclonal Antibody (Cat. No. 701756, 1–2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy chain), Superclonal Recombinant Secondary Antibody, HRP (Cat. No. A27036, 0.4 µg/mL, 1:2,500 dilution).
ACTA2, a protein expressed in smooth muscles, was observed in mouse duodenum compared to mouse brain using Alpha-Smooth Muscle Actin Monoclonal Antibody (1A4) (Cat. No. 14-9760-82).
Figure 2. Immunofluorescence analysis of Her2 was performed using 70% confluent log phase SK-BR-3 cells (panels a–e) and MDA-MB-231 cells (panel f). The cells were fixed with 4% paraformaldehyde for ten minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 1% BSA for one 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, incubated overnight at 4°C, and then labeled with Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor 488 conjugate (Cat. No. A28175) at a dilution of 1:2,000 for 45 minutes at room temperature.
The expression of MAP2, the protein that stabilizes the neurons, is enriched in dendrites which are formed upon the differentiation of the neuroblastoma cell line SH-SY5Y. Signal from MAP2 was observed in differentiated SH-SY5Y cells as compared to the undifferentiated cells, using MAP2 Polyclonal Antibody (Cat. No. PA5-85755) in immunocytochemistry.
Figure 3. Immunofluorescence analysis of MAP2 was performed using SH-SY5Y cells treated with retinoic acid which leads to differentiation (panels a-d) and undifferentiated SH-SY5Y cells (panel e). The cells were fixed, permeabilized and labeled with MAP2 Polyclonal Antibody (Cat. No. PA5-85755) at a dilution of 1:100. Then, the cells were labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Cat. No. A32731) 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 4), 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 4. 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 5), 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 5. Balb/c splenocytes were surface-stained with CD3 (clone 17A2), CD4 (clone GK1.5), and CD8 (clone 53-6.7) antibodies, followed by intracellular staining with Foxp3 (clone FJK-16s) antibody 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.
Secondary antibody specificity validation by relative expression
The differential endogenous expression of antibody isotypes by B-lymphocyte cell lines can also be exploited to validate the specificity of various secondary antibodies. B-lymphocyte cell lines are tested alongside non-antibody producing cell lines to prove that the signal originates from the secondary antibody that is specifically bound to endogenous immunoglobulins.
Relative expression of Human IgG was observed in ARH-77 and IM-9 whole cell lysate; and not in MOLT-4, Jurkat or Raji whole cell lysates. Similarly, the expression was detected in IM-9, ARH-77 conditioned medium (CM) but not in Raji, Jurkat, Molt-4 conditioned medium, using Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, DyLight 755 (Cat. No. SA5-10139) in western blot.
Figure 6. Western blot (non-reducing) was performed using Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, DyLight 755 (Cat. No. SA5-10139). Whole cell lysate (30 µg) of MOLT-4 (Lane 1), Jurkat (Lane 2), Raji (Lane 3), IM-9 (Lane 4), ARH-77 (Lane 5), IM-9 CM (Lane 6), ARH-77 CM (Lane 7), and Jurkat CM (Lane 8) were electrophoresed and were transferred onto a nitrocellulose membrane. The blot was probed with Goat anti-Human IgG Fc Cross-Adsorbed Secondary Antibody, DyLight 755 (1 µg/mL).
Secondary antibody specificity testing can be performed in combination with an advanced validated primary antibody using relative expression. The dynamic range of protein expression will be in synergy with the signal generated, showing a significant difference between the positive/high expressing and negative/low expressing models.
Immunofluorescence analysis in SH-SY5Y cell lines showed a specific signal from Nestin primary antibody when probed with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 800 (Cat. No. A32789). The absence of signal in T-47D (negative expression model for Nestin) and the ‘no primary control’ shows that the signal is from the specific secondary antibody that is bound to the primary antibody.
Figure 7. Immunofluorescence analysis of Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 800 (Cat. No. A32789) was performed using SH-SY5Y (positive model) ad T-47D (negative model) cells stained with Nestin Monoclonal Antibody (10C2), eBioscience (Cat. No. 14-9843-80). The cells were fixed, permeabilized, and blocked. Then, they were labeled with Nestin monoclonal antibody for 3 hours at room temperature, followed by Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 800 at a dilution of 1:2000 (panel a-d). The specificity of the secondary antibody was proved by the absence of signal in T-47D (negative model) (panel e). No primary antibody control has no staining (panel f).
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.
Advanced Verification
Thermo Fisher Scientific is committed to adopting higher validation standards for the Invitrogen antibody portfolio. We have implemented additional specificity tests to help ensure high confidence levels in our products. You can identify the products that have already undergone this testing with the Advanced Verification badge, shown above. This badge can be found in antibody search results and at the top of product webpages. The data supporting the Advanced Verification status can be found in the product specific data galleries. To learn more about our testing standards, please visit Invitrogen Antibody Validation.
*The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.
For Research Use Only. Not for use in diagnostic procedures.
For Research Use Only. Not for use in diagnostic procedures.