Proteins can be expressed in some cell or tissue types but not in others. Antibody target verification can be determined by analyzing the relative expression of these proteins in different cell or tissue types using a variety of applications (immunofluorescence, western blotting, flow cytometry, etc). For example, the transcription factor Nanog is known to be expressed and be present in the nuclei of embryonic stem (ES) cells, induced pluripotent stem (iPS) cells and embryonal carcinoma cell lines (such as NCCIT and NTERA-2). However, Nanog is not present in a variety of other cancer cell lines, such as HeLa cells. The specificity of a Nanog antibody, using positive and negative cell lines, is shown below. The staining of Nanog correlates with the expected staining for Nanog (right, green). Nanog is only present in the nuclei of the NTERA-2 cells, not the HeLa cells (left).

Endogenous Controls-Nanog

Immunofluorescence analysis of Nanog (green) in NTERA-2 and HeLa cells. Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 min at room temperature. Cells were blocked with 1% BSA for 15 min at room temperature. Cells were probed with a Nanog monoclonal antibody (Cat. No. MA1-017) at a dilution of 1:50 for at least 1 hr at room temperature, washed with PBS, and incubated with a DyLight 488–conjugated goat anti–mouse IgG secondary antibody (Cat. No. 35502). F-actin (red) was stained with DyLight 554 phalloidin (Cat. No. 21834) and nuclei (blue) were stained with Hoechst 33342 dye (Cat. No. 62249). Images were taken on a Thermo Scientific ToxInsight image cytometry platform at 20x magnification.


Similar information can also be ascertained using additional applications like western blotting. When testing specificity of the Nanog monoclonal antibody (clone 23D2-3C6) in western blotting, whole cell lysates of 2 cell lines with known Nanog expression were used: NCCIT and NTERA-2. HeLa whole cell lysates were used as a negative control. As shown below, Nanog could be detected in NCCIT and NTERA-2 cell lysates as a prominent band at ~38 kDa and was absent in HeLa lysates (left panel). In addition, nuclear and cytoplasmic fractions were obtained from NCCIT cells and the abundance of Nanog was analyzed using equal amounts of protein from each fraction. As expected, Nanog was detected to be highly abundant in the nuclear fraction of NCCIT cells, further showing good correlation of expected protein localization and antibody mediated detection. Only a minor amount of Nanog was detected in the cytoplasmic fraction, stemming most likely from incomplete separation of the fractions (right panel).

Western blot

Western blot analysis of Nanog in whole cell lysates. Electrophoresis was performed after loading 60 µg of various whole cell lysates and 10 µL of PageRuler Prestained Protein Ladder (Cat. No. 26616) onto a 4–20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hr. The membrane was probed with a Nanog monoclonal antibody (Cat. No. MA1-017) at a dilution of 1:1000 overnight at 4°C on a rocking platform, washed in TBS/0.1% Tween 20, and probed with a goat anti–mouse IgG HRP secondary antibody (Cat. No. 31430) at a dilution of 1:20,000 for 1 hr. Chemiluminescent detection was performed using SuperSignal West Pico substrate (Cat. No. 34078). Note the absence of Nanog in the negative control HeLa cell lysate.

Western blot

Western blot analysis of Nanog in nuclear and cytoplasmic fractions. Electrophoresis was performed after loading 60 µg of NCCIT nuclear and cytoplasmic fractions lysates and 10 µL of PageRuler Prestained Protein Ladder (Cat. No. 26616) onto a 4–20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hr. The membrane was probed with a Nanog monoclonal antibody (Cat. No. MA1-017) at a dilution of 1:1,000 overnight at 4°C on a rocking platform, washed in TBS/0.1% Tween 20, and probed with a goat anti–mouse IgG HRP secondary antibody (Cat. No. 31430) at a dilution of 1:20,000 for 1 hr. Chemiluminescent detection was performed using SuperSignal West Pico substrate (Cat. No. 34078). Nuclear and cytoplasmic fractions were generated using the Thermo Scientific NE-PER kit (Cat. No. 78833).


Similarly, the transcription factor PAX8 is known to be expressed and present in the nuclei of COS7 African green monkey kidney fibroblast cells but not in human 293T cells. The specificity of a PAX8 antibody, using positive and negative cell lines, is shown below. The staining of PAX8 correlates with the expected staining for PAX8 (right, green). PAX8 is only present in the nuclei of COS7 cells, and not in using the 293T cells.

Immunofluorescence analysis of PAX8

Immunofluorescence analysis of PAX8 (green) in 293T (left panel) and COS7 cells (right panel). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 min at room temperature. Cells were blocked with 1% BSA for 15 min at room temperature. Cells were probed with a PAX8 monoclonal antibody (Cat. No. MA1-117) at a dilution of 1:50 for at least 1 hr at room temperature, washed with PBS, and incubated with a DyLight 488–conjugated 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), and nuclei (blue) were stained with Hoechst 33342 dye (Cat. No. 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20x magnification.


To analyze specificity of the PAX8 monoclonal antibody (clone 1F8-3A8) in western blotting, whole cell lysates from 3 negative control cell lines and 4 positive control cell lines expressing varying levels of PAX8 were used. As shown below, PAX8 was not detected in whole cell lysates of ES2 ovarian clear cell carcinoma, MCF7 breast cancer, Jurkat T cell leukemia (left panel), or HeLa (right panel) cell lines. On the other hand, the PAX8 monoclonal antibody (clone 1F8-3A8) detected PAX8 at ~50 kDa in whole cell lysates of Kuramochi high-grade serious ovarian cancer, OVSAHO high-grade serious ovarian carcinoma, SKOV3 ovarian adenocarcinoma, FT246 immortalized fallopian tube epithelium (left panel), and COS7 cells (right panel). All results correlate well with the expected presence or absence of PAX8, demonstrating specificity of the PAX8 monoclonal antibody (clone 1F8-3A8) in western blotting applications.

Western blot analysis of PAX8

Western blot analysis of PAX8. Electrophoresis was performed by loading 20 µg of the indicated whole cell lysates onto an Invitrogen NuPAGE 4–12% Bis-Tris polyacrylamide gel. Proteins were transferred to a nitrocellulose membrane and blocked with 5% milk in PBST for 1 hr. The membrane was probed with a PAX8 monoclonal antibody (Cat. No. MA1-117) at a dilution of 1:500 overnight at 4°C on a rocking platform, washed in PBST, and probed with a goat anti–mouse IgG secondary antibody. Samples were also probed with a beta-actin antibody to control for equal protein loading. Chemiluminescent detection was performed using ECL Plus substrate (Cat. No. 32132). The lysates were: ES2 ovarian clear cell carcinoma, MCF7 breast cancer, Jurkat T cell leukemia, Kuramochi high-grade serious ovarian cancer, OVSAHO high grade serious ovarian carcinoma, SKOV3 ovarian adenocarcinoma, and FT246 immortalized fallopian tube epithelium cells.

Western blot analysis of PAX8

Western blot analysis of PAX8. Electrophoresis was performed by loading 60 µg of SKOV-3, COS7, and negative control HeLa whole cell lysates, and 10 µL of PageRuler Plus Prestained Protein Ladder (Cat. No. 26619) onto a 4–20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hr. The membrane was probed with a PAX8 monoclonal antibody (Cat. No. MA1-117) at a dilution of 1:1,000 overnight at 4°C on a rocking platform, washed in TBS/0.1% Tween 20, and probed with a prediluted (10 µg/mL) HRP-conjugated goat anti–mouse IgG secondary antibody (Cat. No. 32430) at a dilution of 1:500 for 1 hr. Chemiluminescent detection was performed using SuperSignal West Pico substrate (Cat. No. 34080).


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, 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).

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, 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.

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.


*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.