Recombinant Anti-NFAT1 antibody [EPR24658-43] (ab283691)

All lanes : Anti-NFAT1 antibody [EPR24658-43] (ab283691) at 1/1000 dilution

Lane 1 : Wild-type Raji cell lysate
Lane 2 : NFATC2 CRISPR-Cas9 edited Raji cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : SH-SY5Y cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 100 kDa
Observed band size: 100 kDa

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 : Anti-NFAT1 antibody [EPR24658-43] (ab283691) at 1/1000 dilution

Lane 1 : Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate
Lane 2 : Raji (human Burkitts lymphoma B lymphocyte), whole cell lysate
Lane 3 : Daudi (human Burkitts lymphoma lymphoblast), whole cell lysate
Lane 4 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 100 kDa

Blocking and diluting buffer and concentration: 5% NFDM/TBSTThis 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

 

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized 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 (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). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Negative control: Hela (PMID:21078663).

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized 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) (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). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody. Negative control: LNCaP.