Anti-NLRP3 抗体 (ab4207)

False colour image of Western blot: Anti-NLRP3 antibody staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab4207 was shown to bind specifically to NLRP3. A band was observed at 118 kDa in wild-type THP-1 cell lysates with no signal observed at this size in NLRP3 knockout cell line ab280063 (knockout cell lysate ab280122). To generate this image, wild-type and NLRP3 knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in fluorescent western blot (TBS-based) blocking solution 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 then imaged. Secondary antibodies used were Donkey anti-Goat IgG H&L (IRDye^® 800CW) preabsorbed (ab216775) and Donkey anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216778) at 1/20000 dilution.

Immunocytochemistry/Immunofluorescence analysis of paraformaldehyde fixed U937 cells immobilized on ShifixTM coverslip, permeabilized with 0.15% Triton. Incubated with the primary antibody (ab4207) for 1hr at 10 µg/ml, followed by Alexa Fluor^® 488 secondary antibody at 2 µg/ml. Shows cytoplasmic and plasma membrane staining. The nuclear stain is DAPI (blue). Negative control: Unimmunized goat IgG (10 µg/ml) followed by Alexa Fluor^® 488 secondary antibody (2µg/ml).

Flow cytometric analysis of paraformaldehyde fixed U937 cells (blue line), permeabilized with 0.5% Triton. Primary incubation with ab4207 was 1hr (10ug/ml) followed by Alexa Fluor^® 488 secondary antibody (1ug/ml). IgG control: Unimmunized goat IgG (black line) followed by Alexa Fluor^® 488 secondary antibody.

ab4207 staining NLRP3 in the Human White Blood Cells (Mixed Population) by Flow Cytometry. WBC were isolated spinning Blood on Ficoll Gradient after removal of RBC's and permeabilized with 0.1% Triton-X100 in 2% BSA for 15 minutes. The sample was incubated with the primary antibody (1/100 in PBS + 2% BSA in PBS) for 16 hours at 4°C. An Alexa Flour^® 488 Donkey Anti Goat IgG (H+L) (1/250) was used as the secondary antibody.
Gating Strategy: Monocytes

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Immunofluorescence analysis of Human MDM cells, staining CIAS1 / NALP3 with ab4207.
Cells were fixed with paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 5% goat serum for 1 hour at room temp. Samples were incubated with primary antibody (1/200 in 1% BSA in PBS) for 12 hours at 4°C. A Cy5^® conjugated donkey anti-goat polyclonal IgG (1/400) was used as the secondary antibody.

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