Cells were washed twice in ice-cold PBS, scraped in PBS and lysed in a buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% P40, 0.25% sodium deoxycholate, 1 mM EDTA, prepared from a 10X solution (RIPA Lysis Buffer, Millipore) to which was added a cocktail of protease inhibitors (Complete™ Protease inhibitor cocktail, Roche). The samples were incubated for 45 min on ice, then centrifuged for 20 min at 16,000 g at 4°C. The resulting supernatant was removed and the protein concentration of each extract was determined using a DC Protein Assay (Biorad, CA, USA). The proteins were separated by migration in a gel concentration and migration SDS-PAGE and transferred to a PVDF membrane (Immobilon-P Transfer, Millipore) at 4°C for 2 hours at 100 V, blocked using a solution of BSA (Bovine Serum Albumin from 96%, Sigma Aldrich) diluted to 5% in Tris-HCl pH 7.6, 0.1 M NaCl, 0.1% Tween (TBS-T), washed with TBS-Tween 0.1% and immunoblotted with the primary antibody monoclonal anti-PDGFR B (Cell Signaling, catalog#3169, clone#28E1) rabbit diluted by 1/1000 and the rabbit anti with Tag-HA (Santa Cruz, catalog#sc805, clone#Y-11) diluted by 1/1000. The secondary antibody used was anti-rabbit polyclonal IgG HRP (Santa Cruz, catalog#sc2004) diluted by 1/10000.
Immobilon p transfer
Manufactured by Merck Group
Sourced in United States
Immobilon-P transfer membrane is a polyvinylidene difluoride (PVDF) membrane designed for the transfer of proteins and nucleic acids from gel electrophoresis to a solid support for further analysis. It provides efficient and reliable protein and nucleic acid transfer.
Automatically generated - may contain errors
Lab products found in correlation
Ab128568, by Abcam (2 mentions)
Ab214938, by Merck Group (2 mentions)
Westernbright sirus ecl detection kit, by Advansta (2 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Ecl advance western blotting detection kit, by Cytiva (1 mentions)
Anti α tubulin tub antibody, by Merck Group (1 mentions)
Anti rela, by Cell Signaling Technology (1 mentions)
Anti phospho rela ser 536, by Cell Signaling Technology (1 mentions)
Anti smn, by BD (1 mentions)
Anti relb, by Santa Cruz Biotechnology (1 mentions)
Anti creb, by Cell Signaling Technology (1 mentions)
4 protocols using immobilon p transfer
1
Immunoblot Analysis of PDGFR-B Expression
2
Western Blot Analysis of Intestinal Stem Cells
3
Western Blot Analysis of Intestinal Stem Cells
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The following antibodies were used: mouse monoclonal anti-Cpt1a (1:500(sorted), (1:1000crypt lysates), Abcam ab128568), rabbit monoclonal anti-HMGCS2 (1:500(sorted), 1:1000(crypt lysates), Abcam, ab137043), rabbit monoclonal anti-FABP1 (1:1000, CST, 13368), monoclonal rabbit anti-PDK4 (1:1000, Abcam, ab214938), rabbit monoclonal γ-tubulin (1:1000, Sigma-Aldrich, T5198) and monoclonal anti-rabbit total H3 (1:3000, CST 4449S).
Lgr5-eGFPhi ISCs, Lgr5-eGFPlow progenitors, or EpCAM+ cells were sorted directly into 6X Laemmli sample buffer (Alfa Aesar, J61337) and boiled for 5 minutes. Either 10,000 cells were loaded per sorted sample or 10ug of crypt lysates were loaded per sample onto a 4%–12% gradient gel, transferred on to PVDF membrane (Immobilon-P transfer, Millipore, ipvh00010), and analyzed using IgG-HRP antibodies (1:3000, CST, 7076, 7074) and Advansta WesternBright Sirus ECL detection kit (K12043D20).
Lgr5-eGFPhi ISCs, Lgr5-eGFPlow progenitors, or EpCAM+ cells were sorted directly into 6X Laemmli sample buffer (Alfa Aesar, J61337) and boiled for 5 minutes. Either 10,000 cells were loaded per sorted sample or 10ug of crypt lysates were loaded per sample onto a 4%–12% gradient gel, transferred on to PVDF membrane (Immobilon-P transfer, Millipore, ipvh00010), and analyzed using IgG-HRP antibodies (1:3000, CST, 7076, 7074) and Advansta WesternBright Sirus ECL detection kit (K12043D20).
4
Quantification of NF-kB Signaling Pathway
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Western blot analysis was performed as previously described [22] . Cells were rinsed in icecold PBS (pH 7.2) after stimulation or lentiviral transduction. Total cell lysates were collected and resolved in SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride Immobilon-P transfer membrane filters (Millipore, Billerica, MA) using an Amersham Biosciences (Piscataway, NJ, USA) semidry Trans-Blot according to manufacturer instructions. Membranes were blotted with the specific antibodies anti-RelA (1:1000), anti-phosphoRelA (Ser536) (1:1000) or anti-CREB (1:1000) from Cell Signaling Technology; anti-SMN (1:5000) from BD Transduction Laboratories; anti-IKKβ (1:1000) or anti-IKKα (1:1000) from Calbiochem or anti-RelB (1:1000) (C-19) from Santa Cruz, following the instructions of the providers. To control the specific protein content per lane, unless stated otherwise, the membranes were re-probed with a monoclonal anti-α-tubulin (Tub) antibody (1:50,000, Sigma), as described by the provider. Blots were developed using the Super Signal chemiluminescent substrate (Pierce, Rockford, IL, USA) or the ECL Advance Western Blotting detection kit (Amersham Biosciences).
NF-ĸB inhibitor SN50, the inactive control peptide SN50M, and the proteasome inhibitor MG132 were purchased from Calbiochem.
NF-ĸB inhibitor SN50, the inactive control peptide SN50M, and the proteasome inhibitor MG132 were purchased from Calbiochem.
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