Anti-DYRK1A antibody [EPR24132-61] - BSA and Azide free

• WB

Lab

Western blot - Anti-DYRK1A antibody [EPR24132-61] - BSA and Azide free (AB280359)

False colour image of Western blot : Anti-DYRK1A antibody [EPR24132-61] staining at 1/2000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab259869 was shown to bind specifically to DYRK1A. A band was observed at 80/85/90 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in DYRK1A CRISPR-Cas9 edited cell line ab267336 (CRISPR-Cas9 edited cell lysate ab258402). The band observed in the CRISPR-Cas9 edited lysate lane below 80/85/90 kDa is likely to represent a truncated form of DYRK1A. 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 DYRK1A CRISPR-Cas9 edited HEK-293T 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-DYRK1A antibody [EPR24132-61] (<a href='/en-us/products/primary-antibodies/dyrk1a-antibody-epr24132-61-ab259869'>ab259869</a>) at 1/2000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

DYRK1A CRISPR-Cas9 edited HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human DYRK1A knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-dyrk1a-knockout-hek-293t-cell-line-ab267336'>ab267336</a>)

Lane 3:

HT1080 cell lysate at 20 µg

Lane 4:

U-251 MG cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 86 kDa

Observed band size: 80 kDa,85 kDa,90 kDa

false

• WB

Lab

Western blot - Anti-DYRK1A antibody [EPR24132-61] - BSA and Azide free (AB280359)

This data was developed using ab259869, the same antibody clone in a different buffer formulation.

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

This antibody may react with an unidentifiable protein around 30 kDa.

Exposure time : 62 seconds

All lanes:

Western blot - Anti-DYRK1A antibody [EPR24132-61] (<a href='/en-us/products/primary-antibodies/dyrk1a-antibody-epr24132-61-ab259869'>ab259869</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) transfected with scrambled siRNA control whole cell lysate at 20 µg

Lane 2:

DYRK1A knockdown HeLa 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 1/20000 dilution

Predicted band size: 86 kDa

Observed band size: 90 kDa

false

• WB

Lab

Western blot - Anti-DYRK1A antibody [EPR24132-61] - BSA and Azide free (AB280359)

This data was developed using ab259869, the same antibody clone in a different buffer formulation.

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

Lysates were made freshly and used in WB test immediately to minimize protein degradation.

Exposure time : 70 seconds

All lanes:

Western blot - Anti-DYRK1A antibody [EPR24132-61] (<a href='/en-us/products/primary-antibodies/dyrk1a-antibody-epr24132-61-ab259869'>ab259869</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma) 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 1/20000 dilution

Predicted band size: 86 kDa

Observed band size: 90 kDa

false

• WB

Supplier Data

Western blot - Anti-DYRK1A antibody [EPR24132-61] - BSA and Azide free (AB280359)

This data was developed using ab259869, the same antibody clone in a different buffer formulation.

ab259869 was shown to react with DYRK1A in wild-type 5637 cells in Western blot with loss of signal observed in a DYRK1A siRNA knockdown cell line. Cell lysates from wild-type 5637 transfected with either scrambled siRNA or DYRK1A siRNA were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hour before incubation with ab259869 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-DYRK1A antibody [EPR24132-61] (<a href='/en-us/products/primary-antibodies/dyrk1a-antibody-epr24132-61-ab259869'>ab259869</a>) at 1/100 dilution

Lane 1:

Wild-type 5637 transfected with scrambled siRNA control lysate at 60 µg

Lane 2:

5637 transfected with siRNA specifically targeting DYRK1A cell lysate at 60 µg

false

• WB

Lab

Western blot - Anti-DYRK1A antibody [EPR24132-61] - BSA and Azide free (AB280359)

This data was developed using ab259869, the same antibody clone in a different buffer formulation.

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

The expression profile observed is consistent with what has been described in the literature (PMID : 28167836).

Exposure time : 15 seconds

All lanes:

Western blot - Anti-DYRK1A antibody [EPR24132-61] (<a href='/en-us/products/primary-antibodies/dyrk1a-antibody-epr24132-61-ab259869'>ab259869</a>) at 1/1000 dilution

Lane 1:

Mouse E17 brain tissue lysate at 20 µg

Lane 2:

Mouse P1 brain tissue lysate at 20 µg

Lane 3:

Mouse P240 brain tissue 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 1/20000 dilution

Predicted band size: 86 kDa

Observed band size: 90 kDa

false