RIPA lysis buffer (Sigma, Shanghai, China) was applied to extract total proteins. A BCA kit (Sigma) was employed to measure the concentration of protein in the supernatant. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis was used to separate proteins, followed by their electrical transfer to a polyvinylidene fluoride membrane. Post blocking with 5% BSA, membranes were kept overnight at 4 °C alongside diluted primary antibodies: anti-USP11 (1:5000, ab109232, Abcam), anti-NLRP3 (1:1000, ab263899, Abcam, Cambridge, UK), anti-GSDMD-N (1:1000, ab215203, Abcam), anti-caspase-1 (1:1000, ab207802, Abcam), anti-IL-1β (1:1000, ab300501, Abcam), anti-IL-18 (1:1000, ab207323, Abcam), anti-p-IKKβ (1:1000, ab194528, Abcam), anti-IKKβ (1:500, ab32135, Abcam), anti-p-NF-kB (1:1000, ab76302, Abcam), anti-NF-kB (1:1000, ab16502, Abcam), anti-TRAF3 (1:1000, ab155298, Abcam), and anti-β-actin (1:1000, ab8227, Abcam) and then incubation for 1 h by secondary antibody. An ECL chromogenic substrate was used to visualize the bands.
Ab109232
Manufactured by Abcam
Sourced in United Kingdom
Ab109232 is a primary antibody. It is a recombinant monoclonal antibody derived from human tissue.
Automatically generated - may contain errors
Lab products found in correlation
Ab8227, by Abcam (2 mentions)
Bca kit, by Merck Group (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Ab207802, by Abcam (1 mentions)
Ab32135, by Abcam (1 mentions)
Ab207323, by Abcam (1 mentions)
Ab215203, by Abcam (1 mentions)
Ab194528, by Abcam (1 mentions)
Ab76302, by Abcam (1 mentions)
Ab16502, by Abcam (1 mentions)
Ab263899, by Abcam (1 mentions)
Quantity one v4, by Bio-Rad (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Blocking buffer, by Beyotime (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab226977, by Abcam (1 mentions)
Enhanced chemiluminescence, by Solarbio (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Ab205606, by Abcam (1 mentions)
3 protocols using ab109232
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of Protein Expression
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The total protein was extracted from tissue homogenate or cell lysate via the RIPA buffer (Beyotime). After the centrifugation, the supernatant was harvested and quantified through BCA protein assay kit (Beyotime) according to the manufacturer’s instructions. Next, 20 μg of protein was separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked in the blocking buffer (Beyotime) for 1 h, and then incubated with primary antibodies for anti-CyclinD1 (ab226977, 1:300 dilution, Abcam, Cambridge, UK), anti-USP11 (ab109232, 1:3000 dilution, Abcam), or anti-β-actin (ab8227, 1:3000 dilution, Abcam) overnight, followed by incubation of horseradish peroxidase (HRP)-labeled IgG (ab6721, 1:10000 dilution, Abcam) for 2 h. β-actin was used as a loading control. The protein signaling was developed via Enhanced Chemiluminescence (Solarbio). Relative protein level was analyzed using QuantityOne v4.6 (Bio-Rad) and normalized to GAPDH.
3
Protein Interaction Analysis in HEK293T Cells
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Hemagglutinin (HA)-p53, Myc-ubiquitin, and FLAG-USP11 were co-transfected into HEK293T cells for 36 h. The cells were then treated with MG132 (5 μM) for 3 h before collection. The harvested cells were lysed in RAPI buffer: SDS (0.5%, 5 g/L), sodium deoxycholate (0.5%, 5 g/L), Nonidet P-40 (0.5%), NaCl (150 mM), NaF (10 mM), β-glycerophosphate (20 mM), sodium orthovanadate (1 mM), phenylmethylsulfonyl fluoride (1 mM), leupeptin (10 μg/mL), and aprotinin (2 μg/mL). Cells were incubated with anti-HA at 4°C overnight and with protein G agarose beads for another 4 h. The immunoprecipitated proteins were boiled in SDS buffer and measured by western blot assay. All the antibodies used in this assay, including anti-FLAG (ab205606, 1:30), anti-β-actin (ab179467, 1:40), anti-HA (ab18181, 1:50), anti-Myc (ab32072, 1:20), anti-USP11 (ab109232, 1:50), and anti-p53 (ab26, 1:50), were purchased from Abcam.
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