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Cell Treatment Antibody Validation*
Using cell treatments is a common tool used in cell biology to analyze the various biological functions of proteins– from the modulation of signaling pathways to cell cycle and apoptosis. These same biological tools can be applied to verify the specificity of antibodies. Cell treatment testing is one strategy we use to validate Invitrogen antibodies for research use.
Target verification by cell treatment can be based on enrichment, depletion, or translocation of the protein of interest. For example, proteins with dynamic changes in abundance during the cell cycle can be enriched by using specific cell cycle inhibitors. Transcription factors can be activated by cytokine treatment and signaling pathway activation, resulting in translocation to the nucleus. The translocation event can then be more easily monitored with antibodies, compared to the often subtle and dynamic changes in the same cellular compartment.
When analyzing post-translational modification (PTM) states, cell treatments in combination with inhibitors are used to demonstrate that a specific kinase is activated through phosphorylation at a specific site. This is done using phospho-specific antibodies and appropriate controls.
Cell treatment validation data
The following examples demonstrate how we use cell treatment testing to verify target specificity.
In the example below (Figure 1), altered expression of the target protein upon cell treatment demonstrates antibody specificity. Western blot using Phospho-EGFR (Tyr1068) Polyclonal Antibody (Cat. No. PA5-17848) shows increased expression of proteins phosphorylated at the tyrosine residues in A-431 and A549 cell lines upon EGF treatment. Pre-treatment with EGFR-antagonists, Gefitinib and Afatinib, resulted in inhibition of Phospho-EGFR in A-431 and A549 cell lines.
Figure 1. Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of A-431 (Lane 1), A-431 treated with EGF (200 ng/mL for 10 minutes) (Lane 2), A-431 treated with Gefitinib followed by EGF (1 µM for 16 hours, 200 ng/mL for 10 minutes) (Lane 3), A-431 treated with Afatinib followed by EGF (0.5 µM for 6 hours, 200 ng/mL for 10 minutes) (Lane 4), A549 (Lane 5), A549 treated with EGF (200 ng/mL for 10 minutes) (Lane 6), and A549 treated with Afatinib followed by EGF (0.5 µM for 6 hours, 200 ng/mL for 10 minutes) (Lane 7). The blot was probed with Phospho-EGFR (Tyr1068) Rabbit Polyclonal Antibody (Cat. No. PA5-17848, 1:1,000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy chain) Superclonal Recombinant Secondary Antibody, HRP (Cat. No. A27036, 0.25 µg/mL, 1:4,000 dilution).
Cell treatment was also used for verification of antibody specificity for IP applications. In the example below (Figure 2), immunoprecipitation of lysine-acetylated proteins was performed using an acetylated lysine monoclonal antibody on whole cell lysates from cells left untreated or cells treated with trichostatin A (TSA). An acetyl-histone H3 (Lys9) monoclonal antibody was used to detect enrichment of acetylated histone H3 (Lys 9) in response to TSA treatment, confirming the specificity of the acetylated lysine monoclonal antibody for the enrichment of its intended target(s) by immunoprecipitation.
Figure 2. Immunoprecipitation of acetylated Histone H3 (Lys9). Whole cell lysates from cells that were left untreated (DMSO only) or cells treated with 0.3 µM or 3 µM trichostatin A (TSA) for 16 hours. Antigen-antibody complexes were formed by incubating 500 µg of the indicated lysate with 3 µg of an Acetylated Lysine Monoclonal Antibody (1C6) (Cat. No. MA1-2021) overnight on a rocking platform at 4°C. The immune complexes were captured on 50 µL Protein A/G Agarose (Cat. No. 20421), washed extensively, and eluted with 5X Lane Marker Reducing Sample Buffer (Cat. No. 39000). Samples were resolved on a 4–20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBS/0.1% Tween for at least one hour. The membrane was probed with an H3K9ac Monoclonal Antibody (J.924.2) (Cat. No. MA5-11195) at a dilution of 1:1,000 overnight, rotating at 4°C, then washed in TBST and probed with Clean-Blot IP Detection Reagent (Cat. No. 21230) at a dilution of 1:2,000 for at least one hour. Chemiluminescent detection was performed using SuperSignal West Pico substrate (Cat. No. 34087).
Cell treatment was also used to demonstrate specificity in immunofluorescence. Immunofluorescence analysis of Phospho-PDGFRB (Tyr751) using Phospho-PDGFRB (Tyr751) Monoclonal Antibody (C.17.2) (Cat. No. PA5-15192) shows induction of PDGFRB phosphorylation at Tyr751 residue in U-87 MG cell line upon PDGF treatment.
Figure 3. Immunofluorescence analysis of Phospho-PDGFRB (Tyr751) was performed using 70% confluent log phase U-87 MG cells treated with 50 ng of PDGF for 5 minutes. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, and blocked with 1% BSA for one hour at room temperature. The cells were labeled with Phospho-PDGFRB (Tyr751) Monoclonal Antibody (C.17.2) (Cat. No. MA5-15192) at 1:100 dilution in 0.1% BSA, incubated at 4°C overnight, and then labeled with Goat anti-Mouse IgG (H+L) Superclonal Recombinant Secondary Antibody, Alexa Fluor 488 (Cat. No. A28175) at a dilution of 1:2,000 for 45 minutes at room temperature.
Verifying target specificity of Invitrogen antibodies using cell treatments
Invitrogen antibodies that have been verified using cell treatments 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.