PYHIN1 Antibody / Pyrin and HIN domain-containing protein 1 (FY12755)

Immunofluorescent staining of PYHIN1 using anti-PYHIN1 antibody (green) and Alpha Tubulin mAb (red). PYHIN1 was detected in an immunocytochemical section of SiHa cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent for 15 mins. The cells were blocked with 10% goat serum.

Western blot analysis of PYHIN1 using anti-PYHIN1 antibody. Lane 1: human THP-1 whole cell lysates, Lane 2: human Jurkat whole cell lysates, Lane 3: human HEL whole cell lysates, Lane 4: human U251 whole cell lysates, Lane 5: rat spleen tissue lysates, Lane 6: rat C6 whole cell lysates, Lane 7: mouse A20 whole cell lysates. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 150 mA for 50-90 minutes. Blocked the membrane with 5% non-fat milk/TBS for 1.5 hour at RT. The membrane was incubated with rabbit anti-PYHIN1 antibody at 0.5 ug/ml overnight at 4oC, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:5000 for 1.5 hour at RT. The signal was developed using enhanced chemiluminescent. A predominant band is detected at ~55 kDa as expected. In A20 and Jurkat lysates the signal appears as a doublet, consistent with modified or isoform variants of PYHIN1 commonly observed for this protein family.

Immunohistochemical staining of PYHIN1 using anti-PYHIN1 antibody. PYHIN1 was detected in a paraffin-embedded section of human tonsil tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 ug/ml rabbit anti-PYHIN1 antibody overnight at 4oC. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37oC. The tissue section was developed using an HRP secondary and DAB substrate.

Flow Cytometry analysis of ThP-1 cells using anti-PYHIN1 antibody. Overlay histogram showing ThP-1 cells stained with (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-PYHIN1 antibody (1 ug/million cells) for 30 min at 20oC. DyLight 488 conjugated goat anti-rabbit IgG (5-10 ug/million cells) was used as secondary antibody for 30 minutes at 20oC. Isotype control antibody (Green line) was rabbit IgG (1 ug/million cells) used under the same conditions. Unlabelled sample without incubation with primary antibody and secondary antibody (Red line) was used as a blank control.