CXCL2 Recombinant Rabbit Monoclonal Antibody [PSH14-44]

☑ Cell treatment (CT)

Western blot analysis of CXCL2 on different lysates with Rabbit anti-CXCL2 antibody (HA723640) at 1/5,000 dilution.

Lane 1: RAW264.7 cell lysate
Lane 2: RAW264.7 treated with 10μg/mL LPS for 8 hours cell lysate
Lane 3: RAW264.7 cell lysate
Lane 4: RAW264.7 treated with 100ng/mL LPS for 4 hours then add 1μg/mL BFA for 3 hours cell lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 11 kDa
Observed band size: 11 kDa

Exposure time: 1 minute 59 seconds; ECL: K1802;

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 (HA723640) at 1/5,000 dilution was used in primary antibody dilution (K1803) 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 RAW264.7 cells untreated / treated with 200ng/mL LPS for 4 hours then add 1ug/mL BFA for 2 hours labeling CXCL2 with Rabbit anti-CXCL2 antibody (HA723640) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-CXCL2 antibody (HA723640) at 1/100 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 (HA601187, 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)

Immunohistochemical analysis of paraffin-embedded RAW264.7 cells untreated / treated with 200ng/mL LPS for 4 hours then add 1ug/mL BFA for 2 hours with Rabbit anti-CXCL2 antibody (HA723640) at 1/1,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA723640) at 1/1,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

☑ Cell treatment (CT)

Flow cytometric analysis of RAW264.7 cells untreated (left) / treated with 200ng/mL LPS for 4 hours then add 1ug/mL BFA for 2 hours (right) labeling CXCL2.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA723640, 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).

CXCL2 was immunoprecipitated from 0.2 mg RAW264.7 treated with 100ng/mL LPS for 4 hours then add 1μg/mL BFA for 3 hours cell lysate with HA723640 at 2 µg/10 µl beads. Western blot was performed from the immunoprecipitate using HA723640 at 1/2,000 dilution. HRP Conjugated Anti-Rabbit IgG for IP Nano-secondary antibody at 1/5,000 dilution was used for 1 hour at room temperature.

Lane 1: RAW264.7 treated with 100ng/mL LPS for 4 hours then add 1μg/mL BFA for 3 hours cell lysate (input)
Lane 2: HA723640 IP in RAW264.7 treated with 100ng/mL LPS for 4 hours then add 1μg/mL BFA for 3 hours cell lysate
Lane 3: Rabbit IgG instead of HA723640 in RAW264.7 treated with 100ng/mL LPS for 4 hours then add 1μg/mL BFA for 3 hours cell lysate

Blocking/Dilution buffer: 5% NFDM/TBST
Exposure time: 3 minutes; ECL: K1801