Anti-CXCL1/GRO alpha antibody [EPR19892]

• IP

Lab

Immunoprecipitation - Anti-CXCL1/GRO alpha antibody [EPR19892] (AB206411)

11 kDa CXCL1/GRO alpha was immunoprecipitated from 0.35mg of HUVEC (human umbilical vein endothelial cell) whole cell lysate with ab206411 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab206411 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution. Lane 1 (Input) : HUVEC treated with 0.5 μg/ml LPS for 4 hours, then added 300 ng/ml BFA for another 20 hours whole cell lysate 10 μg. Lane 2 (+) : ab206411-IP in HUVEC treated with 0.5 μg/ml LPS for 4 hours, then added 300 ng/ml BFA for another 20 hours whole cell lysate. Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab206411 in HUVEC treated as above whole cell lysate. Blocking buffer and concentration : 5% NFDM/TBST. Diluting buffer and concentration : 5% NFDM/TBST. Exposure time : 180 seconds.

All lanes:

Immunoprecipitation - Anti-CXCL1/GRO alpha antibody [EPR19892] (ab206411)

Lane 1:

HUVEC (human umbilical vein endothelial cell) treated with 0.5 μg/ml LPS for 4 hours, then added 300 ng/ml BFA for another 20 hours whole cell lysate at 10 µg

Lane 2:

HUVEC (human umbilical vein endothelial cell) treated with 0.5 μg/ml LPS for 4 hours, then added 300 ng/ml BFA for another 20 hours whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 11 kDa

Observed band size: 11 kDa

false

• WB

Supplier Data

Western blot - Anti-CXCL1/GRO alpha antibody [EPR19892] (AB206411)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CXCL1/GRO alpha antibody [EPR19892] (ab206411) at 1/100 dilution

All lanes:

Human fetal lung lysate at 10 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: 11 kDa

Observed band size: 11 kDa

false

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-CXCL1/GRO alpha antibody [EPR19892] (AB206411)

Blocking/Dilution buffer : 5% NFDM/TBST.

Human GRO beta fragment recombinant protein contain aa35-107 with a GST-Tag. Human GRO gamma fragment recombinant protein contain aa35-107 with a His-Tag®. These two protein were made in house.

All lanes:

Western blot - Anti-CXCL1/GRO alpha antibody [EPR19892] (ab206411) at 1/1000 dilution

Lane 1:

Human CXCL1/GRO alpha active protein at 0.01 µg

Lane 2:

Human GRO beta fragment recombinant protein at 0.01 µg

Lane 3:

Human GRO gamma fragment recombinant protein at 0.01 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: 11 kDa

Observed band size: 10 kDa,36 kDa

false

Exposure time: 3min

• WB

Lab

Western blot - Anti-CXCL1/GRO alpha antibody [EPR19892] (AB206411)

False colour image of Western blot : Anti-CXCL1/GRO alpha antibody [EPR19892] staining at 1/100 dilution, shown in black; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab206411 was shown to bind specifically to CXCL1/GRO alpha. A band was observed at 8 kDa in treated wild-type HeLa cell lysates with no signal observed at this size in CXCL1 knockout cell line ab261774 (knockout cell lysate ab257079). To generate this image, wild-type and CXCL1 knockout HeLa 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 before development with Optiblot (ECL reagent ab133456) and imaged with 20 minutes exposure time. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and HRP conjugated Goat anti-Mouse (H+L) at 1/20000 dilution.

All lanes:

Western blot - Anti-CXCL1/GRO alpha antibody [EPR19892] (ab206411) at 1/100 dilution

Lane 1:

Wild-type HeLa Treated TNF alpha (5 ng/mL, 6 h) + BFA (5 ug/mL, 6 h) cell lysate at 20 µg

Lane 2:

CXCL1 knockout HeLa Treated TNF alpha (5 ng/mL, 6 h) + BFA (5 ug/mL, 6 h) cell lysate at 20 µg

Lane 2:

Western blot - Human CXCL1 (GRO alpha) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-cxcl1-gro-alpha-knockout-hela-cell-line-ab261774'>ab261774</a>)

Predicted band size: 11 kDa

Observed band size: 8 kDa

false