Anti-NFAT1 antibody [EPR24658-43] - BSA and Azide free

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-NFAT1 antibody [EPR24658-43] - BSA and Azide free (AB283720)

This data was developed using ab283691, the same antibody clone in a different buffer formulation.Flow cytometric analysis of HeLa (human cervix adenocarcinoma epithelial cell, Left) / Ramos (Human Burkitt's lymphoma B lymphocyte, Right) cells labelling NFAT1 with ab283691 at 1/50 dilution (1ug)/ red compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Negative control : Hela (PMID : 21078663).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-NFAT1 antibody [EPR24658-43] - BSA and Azide free (AB283720)

This data was developed using ab283691, the same antibody clone in a different buffer formulation.Flow cytometric analysis of LNCaP (Human prostate carcinoma epithelial cell, Left) / Jurkat (Human T cell leukemia T lymphocyte, Right) cells labelling NFAT1 with ab283691 at 1/50 dilution (1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Negative control : LNCaP.

• WB

Lab

Western blot - Anti-NFAT1 antibody [EPR24658-43] - BSA and Azide free (AB283720)

This data was developed using ab283691, the same antibody clone in a different buffer formulation.

False colour image of Western blot : Anti-NFAT1 antibody [EPR24658-43] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab283691 was shown to bind specifically to NFAT1. A band was observed at 100 kDa in wild-type Raji cell lysates with no signal observed at this size in NFATC2 CRISPR-Cas9 edited cell line ab280906 (CRISPR-Cas9 edited cell lysate ab282940). The band observed in the CRISPR-Cas9 edited lysate lane below 100 kDa is likely to represent a truncated form of NFAT1. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and NFATC2 CRISPR-Cas9 edited Raji cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-NFAT1 antibody [EPR24658-43] (<a href='/en-us/products/primary-antibodies/nfat1-antibody-epr24658-43-ab283691'>ab283691</a>) at 1/1000 dilution

Lane 1:

Wild-type Raji cell lysate at 20 µg

Lane 2:

NFATC2 CRISPR-Cas9 edited Raji cell lysate at 20 µg

Lane 2:

Western blot - Human NFATC2 knockout Raji cell line (<a href='/en-us/products/cell-lines/human-nfatc2-knockout-raji-cell-line-ab280906'>ab280906</a>)

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

SH-SY5Y cell lysate at 20 µg

Predicted band size: 100 kDa

Observed band size: 100 kDa

false

• WB

Lab

Western blot - Anti-NFAT1 antibody [EPR24658-43] - BSA and Azide free (AB283720)

This data was developed using ab283691, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : This blot was developed using a higher-sensitivity ECL substrate.

The molecular weight observed is consistent with what has been described in the literature (PMID : 21078663, PMID : 25696812).

Negative control : Hela (PMID : 21078663)

Exposure time : 3 minutes

All lanes:

Western blot - Anti-NFAT1 antibody [EPR24658-43] (<a href='/en-us/products/primary-antibodies/nfat1-antibody-epr24658-43-ab283691'>ab283691</a>) at 1/1000 dilution

Lane 1:

Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate at 20 µg

Lane 2:

Raji (human Burkitts lymphoma B lymphocyte), whole cell lysate at 20 µg

Lane 3:

Daudi (human Burkitts lymphoma lymphoblast), whole cell lysate at 20 µg

Lane 4:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 100 kDa

false