Anti-NEK9 antibody [EP7361] - BSA and Azide free

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-NEK9 antibody [EP7361] - BSA and Azide free (AB248844)

This data was developed using ab138488, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of HeLa cells labelling NEK9 with unpurified ab138488 at 1/250 dilution.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-NEK9 antibody [EP7361] - BSA and Azide free (AB248844)

This data was developed using ab138488, the same antibody clone in a different buffer formulation.

Intracellular Flow Cytometry analysis of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Sonic Hedgehog with purified ab53281 at 1/20 dilution (10 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - cells without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NEK9 antibody [EP7361] - BSA and Azide free (AB248844)

This data was developed using ab138488, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labelling NEK9 with unpurified ab138488 at 1/50 dilution.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-NEK9 antibody [EP7361] - BSA and Azide free (AB248844)

This data was developed using ab138488, the same antibody clone in a different buffer formulation.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human testis tissue sections labeling NEK9 with Purified ab138488 at 1 : 100 dilution (3.84 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1 : 0 dilution. PBS instead of the primary antibody was used as the negative control.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NEK9 antibody [EP7361] - BSA and Azide free (AB248844)

This data was developed using ab138488, the same antibody clone in a different buffer formulation.

Immunocytochemistry/ Immunofluorescence analysis of NIH/3T3 (Mouse embryonic fibroblast) cells labeling NEK9 with Purified ab138488 at 1 : 100 dilution (3.8μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1 : 200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1 : 1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• WB

Lab

Western blot - Anti-NEK9 antibody [EP7361] - BSA and Azide free (AB248844)

This data was developed using ab138488, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-NEK9 antibody [EP7361] (<a href='/en-us/products/primary-antibodies/nek9-antibody-ep7361-ab138488'>ab138488</a>) at 1/5000 dilution

Lane 1:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysate at 15 µg

Lane 2:

THP-1 (Human monocytic leukemia monocyte) whole cell lysate at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 107 kDa

Observed band size: 120 kDa

false

• WB

Lab

Western blot - Anti-NEK9 antibody [EP7361] - BSA and Azide free (AB248844)

This data was developed using the same antibody clone in a different buffer formulation (ab138488).

Lanes 1 - 4 : Merged signal (red and green). Green - ab138488 observed at 120 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A]) observed at 55kDa.

ab138488 was shown to react with NEK9 in A431 wild-type cells in western blot. Loss of signal was observed when NEK9 knockout sample was used. Wild-type and NEK9 knockout A431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab138488 and ab7291 (Mouse anti-Alpha Tubulin [DM1A]) overnight at 4°C at a 1 in 1000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-NEK9 antibody [EP7361] (<a href='/en-us/products/primary-antibodies/nek9-antibody-ep7361-ab138488'>ab138488</a>) at 1/1000 dilution

Lane 1:

Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

NEK9 knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human NEK9 knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-nek9-knockout-a-431-cell-line-ab270474'>ab270474</a>)

Lane 3:

Hep G2 (Human liver hepatocellular carcinoma cell line) whole cell lysate at 20 µg

Lane 4:

MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg

Predicted band size: 107 kDa

Observed band size: 120 kDa

false

• WB

Lab

Western blot - Anti-NEK9 antibody [EP7361] - BSA and Azide free (AB248844)

This data was developed using ab138488, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-NEK9 antibody [EP7361] (<a href='/en-us/products/primary-antibodies/nek9-antibody-ep7361-ab138488'>ab138488</a>) at 1000 Cells

All lanes:

Rat spleen lysates at 15 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 107 kDa

Observed band size: 120 kDa

false

• WB

Unknown

Western blot - Anti-NEK9 antibody [EP7361] - BSA and Azide free (AB248844)

This data was developed using ab138488, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-NEK9 antibody [EP7361] (<a href='/en-us/products/primary-antibodies/nek9-antibody-ep7361-ab138488'>ab138488</a>) at 1/1000 dilution

Lane 1:

NIH3T3 cell lysate at 10 µg

Lane 2:

HepG2 cell lysate at 10 µg

Lane 3:

HeLa cell lysate at 10 µg

Lane 4:

THP1 cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 107 kDa

false