Whole protein lysates were isolated from colon biopsies, HCT116, Caco2 and HT-29 cells using cell lysis buffer (Cell Signaling, Cat. No. 9803) supplemented with 1 mM PMSF (Cell Signaling, Cat. No. 8553) and centrifuged at 14,000 rpm for 20 min. MiniProtean-TGX gels (4–15% polyacrylamide; Bio-Rad, Hercules, CA, USA) were used for protein separation. Separated proteins were blotted on a PVDF (Bio-Rad, Cat. No. 1704272) or nitrocellulose membrane (Bio-Rad, Cat. No. 1704270). Membranes were incubated with the following primary antibodies: MALT1 (Cell Signaling, Cat. No. 2494), ERK (Cell Signaling, Cat. No. 9102), p100/p52 (Cell Signaling, Cat. No. 4882), claudin-4 (Invitrogen, Cat. No. 36-4800). Anti-rabbit IgG, HRP-linked antibody (Cell Signaling, Cat. No. 7074) was used as a secondary antibody. Western Lightning Plus Chemiluminescent Substrate (PerkinElmer, Waltham, MA, USA, Cat. No. NEL105001EA) was used for detection. Densitometric analysis was performed using the Image Lab Software (Bio-Rad).
Western lightning plus chemiluminescent substrate
Manufactured by PerkinElmer
Sourced in United States, Germany
The Western Lightning Plus Chemiluminescent Substrate is a reagent used in Western blot analysis to detect and quantify proteins. It generates a chemiluminescent signal upon interaction with the enzyme-labeled secondary antibody, allowing for the visualization and analysis of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Cell lysis buffer, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
Claudin 4, by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Cell Signaling Technology (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Malt1, by Cell Signaling Technology (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Non fat dry milk, by Bio-Rad (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Novex wedgewell 4 20 tris glycine gel, by Thermo Fisher Scientific (1 mentions)
Hyblot cl autoradiography film, by Thomas Scientific (1 mentions)
Bovine serum albumin (bsa), by Bio-Rad (1 mentions)
Nonfat dry milk, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Flotillin 2, by Cell Signaling Technology (1 mentions)
Iκbα 4812, by Cell Signaling Technology (1 mentions)
Hsp90, by Cell Signaling Technology (1 mentions)
3 protocols using western lightning plus chemiluminescent substrate
1
Western Blot Analysis of Colon Cell Proteins
2
Western Blot of Membrane Vesicles
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Membrane vesicle preparations (10 μg protein per lane) from HEK293 and BSEP-EYFP cells were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Blot membranes were cut into segments and blocked for 1 h in TBS-T containing 10% (w/v) non-fat dried milk. Segments were probed with human serum samples (1:1,000 in block) for 1 h followed by goat antihuman IgG-HRP conjugate (1:10,000 in TBS-T) for 1 h before detection using Western Lightning Plus chemiluminescent substrate (PerkinElmer, Rodgau, Germany) in a ChemiDoc MP Imaging System (Biorad, Feldkirchen, Germany).
3
Western Blot Protein Analysis
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For western blot analysis, equal amounts of proteins were separated using Novex WedgeWell 4-20%, Tris-Glycine gels (Invitrogen, Thermo Fisher Scientific). Proteins were transferred onto PVDF membranes (Thermo Fisher). Membranes were blocked with a solution of 5% non-fat dry milk (Bio-Rad Laboratories) in Tris Buffered Saline -0.5% Tween-20 (TBS-T), then incubated with primary antibody in a solution of 5% bovine serum albumin (BSA) (Bio-Rad Laboratories) in TBS-T overnight at 4 °C. Membranes were then washed in TBS-T solution, and incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (anti-mouse, #7076, or anti-rabbit, #7074, Cell Signalling Technology, Inc.) in 5% non-fat dry milk in TBS-T, for 1 h at room temperature. After additional washes, membranes were incubated with a chemiluminescent detection reagent (Western Lightning Plus, Chemiluminescent Substrate, PerkinElmer Inc.), and imaged on HyBlot CL Autoradiography Film (Thomas Scientific), and the film was developed using developer and fixer solutions (Merry X-Ray Imaging, Inc.). The following primary antibodies were employed: HSP90 (#4877), IκBα (#4812), NAMPT (#6122), β-actin (#3700) and Flotillin-2 (#3436), all from Cell Signaling Technology, Inc. Quantification of Western blots was performed using the ImageJ software (103) .
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