Using cell treatments is a common tool used in cell biology to analyze various biologic 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 phosphospecific antibodies and appropriate controls. 

Cell treatment validation data

In the below example, cell treatment with nocodazole was used along with siRNA knockdown to verify target specificity. The serine/threonine protein kinase PLK1, also known as polo-like kinase 1, is enriched during G2/M cell cycle arrest. PLK1 is shown in whole cell lysates of untreated or nocodazole-treated U2OS cells. A nontargeting control siRNA or PLK1 siRNA was also used to verify antibody specificity. GAPDH was used as a loading control.

Fig. 1. Western blot analysis of PLK1. Electrophoresis was performed by loading 25 µg of untreated or nocodazole-treated (100 nM, 48 hr) U2OS lysate from nontargeting control or PLK1 siRNA–transfected U2OS cells onto a 4–20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% milk/0.1% Tween 20  for at least 1 hr. Membranes were probed with PLK1 mouse monoclonal antibody (Cat. No. MA1-848) at a dilution of 1:1,000 overnight at 4°C on a rocking platform. Membranes were washed in TBS/0.1% Tween 20 and probed with an anti–mouse IgG HRP secondary antibody (Cat. No. 31430) at a dilution of 1:20,000 for at least 1 hr. Membranes were washed and chemiluminescent detection was performed using SuperSignal West Dura substrate (Cat. No. 34075).


Cell treatment was also used for verification of antibody specificity for IP applications. In the example below, immunoprecipitation of lysine-acetylated proteins was performed using 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. 

Fig. 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 hr. Antigen–antibody complexes were formed by incubating 500 µg of the indicated lysate with 3 µg of an acetyl lysine monoclonal antibody (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 1 hr. The membrane was probed with an acetyl-histone H3 (Lys9) monoclonal antibody (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 1 hr. Chemiluminescent detection was performed using SuperSignal West Pico substrate (Cat. No. 34087).


Cell treatment was also used to verify specificity of the acetylated lysine monoclonal antibody (left panel) and phospho-ATM (Ser1981) monoclonal antibody (right panel) in immunofluorescence. In the absence of treatment, HeLa cells show only minor levels of staining in the nucleus. In response to TSA (left panel) or camptothecin (right panel), nuclear staining of target proteins is greatly increased. These results correlate well with testing results obtained in other applications, such as western blotting, thus confirming antibody specificity.

Immunofluorescence analysis of lysine acetylated proteins
Immunofluorescence analysis of ATM phosphorylated

Fig. 3. Immunofluorescence analysis of lysine acetylated proteins (green) in HeLa cells left untreated (left panels) or treated with 0.3 µM trichostatin A for 16 hr (right panels). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 min at room temperature and blocked with 1% BSA for 15 min at room temperature. Cells were probed with an acetyl lysine monoclonal antibody (Cat. No. MA1-2021) at a dilution of 1:100 for 1 hr at room temperature, washed with PBS, and incubated with DyLight 488 goat anti–mouse IgG secondary antibody (Cat. No. 35502) at a dilution of 1:400 for 30 min at room temperature. F-actin (red) was stained with DyLight 554 phalloidin (Cat. No. 21834). A merged image with nuclear staining (blue) using Hoechst 33342 dye (Cat. No. 62249) is shown on bottom panels. Images were taken on a Thermo Scientific ArrayScan imaging system at 20x magnification.

Fig. 4. Immunofluorescence analysis of ATM phosphorylated on pSer1981 (green) in HeLa cells left untreated (left panels) or treated with 10 µM camptothecin for 4 hr (right panels). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 min at room temperature and blocked with 1% BSA for 15 min at room temperature. Cells were probed with a phospho-ATM (pSer1981) monoclonal antibody (Cat. No. MA1-2020), at a dilution of 1:1,000 for at least 1 hr at room temperature, washed with PBS, and incubated with DyLight 488 goat anti–mouse IgG secondary antibody (Cat. No. 35502) at a dilution of 1:400 for 30 min at room temperature. F-Actin (red) was stained with DyLight 554 phalloidin (Cat. No. 21834) (top panels). Merged images with nuclear staining (blue) using Hoechst 33342 dye (Cat. No. 62249) are shown in the bottom panels. Images were taken on a Thermo Scientific ArrayScan imaging system at 20x magnification.


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. 

*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.