TNFRSF14 Recombinant Rabbit Monoclonal Antibody [PSH06-02]

☑ Cell treatment (CT),Relative expression (RE)

Western blot analysis of TNFRSF14 on different lysates with Rabbit anti-TNFRSF14 antibody (HA722501) at 1/1,000 dilution.

Lane 1: SK-MEL-28 cell lysate
Lane 2: SK-MEL-28 cell lysate treated with deglycosylation
Lane 3: A549 cell lysate (negative)

Lysates/proteins at 20 µg/Lane.

Predicted band size: 30 kDa
Observed band size: 40-50 kDa

Exposure time: 20 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 (HA722501) 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.

☑ Relative expression (RE)

Immunocytochemistry analysis of SK-MEL-28 (positive) and A549 (negative) labeling TNFRSF14 with Rabbit anti-TNFRSF14 antibody (HA722501) at 1/50 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-TNFRSF14 antibody (HA722501) at 1/50 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.

☑ Relative expression (RE)

Flow cytometric analysis of SK-MEL-28 (left, positive) and A549 (right, negative) cells labeling TNFRSF14.

Cells were washed twice with cold PBS and resuspend. Then stained with the primary antibody (HA722501, 1μg/mL) (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).