CXCL5 Recombinant Rabbit Monoclonal Antibody [JE53-78]

☑ Cell treatment (CT)

Western blot analysis of CXCL5 on different lysates with Rabbit anti-CXCL5 antibody (HA722561) at 1/1,000 dilution.

Lane 1: A549 cell lysate
Lane 2: A549 starved overnight treated with 10ng/mL TNF-alpha and 10nM PMA for 24 hours then add 300ng/mL BFA for 3 hours cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 12 kDa
Observed band size: 8 kDa

Exposure time: 16 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA722561) at 1/1,000 dilution was used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

☑ Cell treatment (CT)

Immunocytochemistry analysis of A549 cells starved overnight treated with 10ng/mL TNF-alpha and 10nM PMA for 24 hours then add 300ng/mL BFA for 3 hours labeling CXCL5 with Rabbit anti-CXCL5 antibody (HA722561) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-CXCL5 antibody (HA722561) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

☑ Cell treatment (CT)

Flow cytometric analysis of HeLa cells starved overnight treated with 10ng/mL TNF-alpha and 10nM PMA for 24 hours then add 300ng/mL BFA for 3 hours labeling CXCL5.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA722561, 1/1,000) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).

☑ Cell treatment (CT)

CXCL5 was immunoprecipitated from 0.2 mg A549 starved overnight treated with 10ng/mL TNF-alpha and 10nM PMA for 24 hours then add 300ng/mL BFA for 3 hours cell lysate with HA722561 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA722561 at 1/1,000 dilution. Anti-Rabbit IgG for IP Nano-secondary antibody (NBI01H) at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: A549 starved overnight treated with 10ng/mL TNF-alpha and 10nM PMA for 24 hours then add 300ng/mL BFA for 3 hours cell lysate (input)
Lane 2: HA722561 IP in A549 starved overnight treated with 10ng/mL TNF-alpha and 10nM PMA for 24 hours then add 300ng/mL BFA for 3 hours cell lysate
Lane 3: Rabbit IgG instead of HA722561 in A549 starved overnight treated with 10ng/mL TNF-alpha and 10nM PMA for 24 hours then add 300ng/mL BFA for 3 hours cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 38 seconds; ECL: K1801