Anti-NFAT1 antibody [EPR24658-149]

• WB

Lab

Western blot - Anti-NFAT1 antibody [EPR24658-149] (AB283649)

False colour image of Western blot : Anti-NFAT1 antibody [EPR24658-149] 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, ab283649 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.

Lanes 1 and 3:

Western blot - Anti-NFAT1 antibody [EPR24658-149] - BSA and Azide free (<a href='/en-us/products/primary-antibodies/nfat1-antibody-epr24658-149-bsa-and-azide-free-ab283659'>ab283659</a>) at 1/1000 dilution

Lanes 2 and 4:

Western blot - Anti-NFAT1 antibody [EPR24658-149] (ab283649) 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 [EPR24658-149] (AB283649)

False colour image of Western blot : Anti-NFAT1 antibody [EPR24658-149] 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, ab283649 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 knockout cell line ab280906 (knockout cell lysate ab282940). The band observed in the knockout 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 knockout 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 [EPR24658-149] (ab283649) at 1/1000 dilution

Lane 1:

Wild-type Raji cell lysate at 20 µg

Lane 2:

NFATC2 knockout Raji cell lysate at 20 µg

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 [EPR24658-149] (AB283649)

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

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 : Lane 1, 2, 4, 5 : 3 minutes ; Lane 3 : 92 seconds

All lanes:

Western blot - Anti-NFAT1 antibody [EPR24658-149] (ab283649) 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:

EL4 (mouse lymphoma T lymphocyte), whole cell lysate at 20 µg

Lane 5:

HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate at 20 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 1/100000 dilution

Predicted band size: 100 kDa

Observed band size: 100 kDa,140 kDa

false