Anti-LKB1 antibody [EPR19379]

• IP

Supplier Data

Immunoprecipitation - Anti-LKB1 antibody [EPR19379] (AB199970)

LKB1 was immunoprecipitated from 1 mg of Human testis lysate with ab199970 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab199970 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1 : Human testis lysate, 10 μg (Input).

Lane 2 : ab199970 IP in Human testis lysate .

Lane 3 : Rabbit IgG,monoclonal [EPR25A]-Isotype Control (ab172730) instead of ab199970 in Human testis lysate .

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 minutes.

LKB1 isoforms : PMID 18854309.

All lanes:

Immunoprecipitation - Anti-LKB1 antibody [EPR19379] (ab199970)

Predicted band size: 48 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-LKB1 antibody [EPR19379] (AB199970)

LKB1 was immunoprecipitated from 1 mg of K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate with ab199970 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab199970 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : K562 whole cell lysate, 10 μg (Input).

Lane 2 : ab199970 IP in K562 whole cell lysate.

Lane 3 : Rabbit IgG,monoclonal [EPR25A]-Isotype Control (ab172730) instead of ab199970 in K562 whole cell lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 minutes.

LKB1 isoforms : PMID 18854309.

All lanes:

Immunoprecipitation - Anti-LKB1 antibody [EPR19379] (ab199970)

Predicted band size: 48 kDa

false

• WB

Lab

Western blot - Anti-LKB1 antibody [EPR19379] (AB199970)

Western blot : Anti-STK11 antibody [EPR19379] (ab199970) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab199970 was shown to bind specifically to STK11. A band was observed at 50-60 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in STK11 knockout cell line. To generate this image, wild-type and STK11 knockout HCT 116 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-LKB1 antibody [EPR19379] (ab199970) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human STK11 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-stk11-knockout-hct116-cell-line-ab286626'>ab286626</a>)

Lane 2:

STK11 knockout HCT 116 cell lysate at 20 µg

Lane 3:

Wild-type HEK-293T ab255553 cell lysate at 20 µg

Lane 4:

STK11 knockout HEK-293T ab261047 cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 50-60 kDa

false

• WB

Supplier Data

Western blot - Anti-LKB1 antibody [EPR19379] (AB199970)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-LKB1 antibody [EPR19379] (ab199970) at 1/1000 dilution

Lane 1:

Human brain lysate at 10 µg

Lane 2:

Human fetal heart lysate at 10 µg

Lane 3:

Human fetal kidney lysate at 10 µg

Lane 4:

Human fetal testis lysate at 10 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Predicted band size: 48 kDa

Observed band size: 50 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-LKB1 antibody [EPR19379] (AB199970)

Lanes 1- 4 : Merged signal (red and green). Green - ab199970 observed at 55 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab199970 was shown to react with LKB1 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266647 (knockout cell lysate ab257712) was used. Wild-type HEK-293T and STK11 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab199970 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-LKB1 antibody [EPR19379] (ab199970) at 1/1000 dilution

Lane 1:

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

Lane 2:

STK11 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human STK11 (LKB1) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-stk11-lkb1-knockout-hek-293t-cell-line-ab266647'>ab266647</a>)

Lane 3:

Hu testis cell lysate at 20 µg

Lane 4:

HeLa cell lysate at 20 µg

Predicted band size: 48 kDa

Observed band size: 55 kDa

false

• WB

Supplier Data

Western blot - Anti-LKB1 antibody [EPR19379] (AB199970)

Blocking/Dilution buffer : 5% NFDM/TBST.

Negative control : HeLa (PMID 11297520; PMID24086281; PMID 19340305).

All lanes:

Western blot - Anti-LKB1 antibody [EPR19379] (ab199970) at 1/1000 dilution

Lane 1:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 2:

K562 (Human chronic myelogenous leukemia cell line from bone marrow) whole cell lysate at 20 µg

Lane 3:

HeLa (Human epithelial cell line from cervix adenocarcinoma) 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: 155 kDa,48 kDa,67 kDa

Observed band size: 100 kDa,180 kDa,50 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-LKB1 antibody [EPR19379] (AB199970)

Lane 1 : Wild-type HAP1 whole cell lysate (20 μg)
Lane 2 : STK11 knockout HAP1 whole cell lysate (20 μg)
Lane 3 : Hu Testis whole cell lysate (20 μg)

Lanes 1 - 3 : Merged signal (red and green). Green - ab199970 observed at 50 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab199970 was shown to specifically react with STK11 in wild-type HAP1 cells. No band was observed when STK11 knockout samples were examined. Wild-type and STK11 knockout samples were subjected to SDS-PAGE. ab199970 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/10,000 dilution respectively. Blots were developed 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 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-LKB1 antibody [EPR19379] (ab199970)

Predicted band size: 48 kDa

false