The spleen tissue was homogenized in Pierce RIPA lysis buffer (Thermo Scientific, Chicago, IL) containing 25 mmol/L Tris-HCl pH 7.6, 150 mmol/L NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail (Sigma Aldrich, St. Louis, MO, USA). Protein samples were resolved on SDS polyacrylamide gel, transferred to PVDF membrane, blocked using 5% (w/v) nonfat dried milk or 5% BSA in Tris-buffered saline containing 25 mmol/L Tris-HCl (pH 7. 4), 0.13 mol/L NaCl, 0.0027 mol/L KCl and 0.1% Tween 20 for 1 h at room temperature (RT) and then incubated with respective antibodies overnight at 4°C or for 1 h at RT. The membranes were probed with antibodies to GAPDH, VDAC, HMGB1, heat shock protein 60 (hsp60) (Thermo Scientific). The membranes were washed and incubated with horseradish peroxidase conjugated secondary antibody to mouse IgG or rabbit IgG (Cell Signalling) followed by enhanced western lightning plus-ECL (PerkinElmer). The protein bands were detected by autoradiography and quantitated by densitometry using the ImageJ software (NIH, Rockville, MD).
Enhanced western lightning plus ecl
Manufactured by PerkinElmer
Sourced in United States
Enhanced Western Lightning® Plus-ECL is a chemiluminescent detection reagent designed for western blotting. It provides a bright, stable signal for the detection of low-abundance proteins.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (2 mentions)
Radioimmunoprecipitation assay buffer, by Cell Signaling Technology (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Pierce ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Hmgb1, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Anti brd4, by Abcam (1 mentions)
Ab76013, by Abcam (1 mentions)
Ab40876, by Abcam (1 mentions)
Mouse anti clathrin heavy chain, by BD (1 mentions)
4 protocols using enhanced western lightning plus ecl
1
Western Blot Analysis of Spleen Tissue
2
Immunoblotting Protocol for Protein Expression
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Immunoblotting was performed as previously described [19 (link),20 (link)]. Briefly, the cells were lysed in a RIPA lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and supernatants were collected by centrifugation at 13,000 rpm for 15 min at 4 °C. Protein quantification in each sample was performed using a BCA™ protein assay kit (Pierce, Rockford, IL, USA) with bovine serum albumin (BSA) as a standard. Equal amounts of proteins were denatured and fractionated by SDS–PAGE, and the proteins were transferred onto nitrocellulose membranes. Nonspecific binding sites were blocked by incubating the membranes in 5% nonfat milk in TBS containing 0.1% Tween-20. The membranes were probed with appropriate primary antibodies overnight at 4 °C followed by appropriate horseradish peroxidase (HRP) conjugated secondary antibody for 1 h at room temperature. Protein expression was detected by enhanced Western Lightning Plus-ECL, enhanced chemiluminescence substrate (PerkinElmer, Shelton, CT, USA). Images were acquired on a Bio-Rad ChemiDoc system by using Image lab software. Expression of GAPDH was used as an internal control for total protein-loading.
3
Brd4 Protein Quantification by Western Blot
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Undifferentiated 20D-17 cells were seeded at a density of 1.0 × 105 cells in a 10 cm dish. Two days after seeding, 0.2 μM JQ1 was added for 24 hours. Cultured cells were treated according to the conditions indicated above; then, proteins were extracted using radioimmunoprecipitation assay buffer (#9806, Cell Signaling Technology, Inc., Danvers, MA). Total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting. Western blotting was performed with the ChemiDoc™ Imaging System (Bio-Rad Laboratories, Inc.). Quantification of protein signals was performed using Image Lab Software, according to the manufacturer's protocol (Bio-Rad Laboratories, Inc.). The primary antibody was anti-Brd4 (dilution 1 : 2000) (Abcam, ab128874). Secondary antibodies were anti-rabbit IgG horseradish peroxidase- (HRP-) conjugated (1 : 2000, Cell Signaling Technology, Inc., 7074). Proteins were visualized using enhanced Western Lightning® Plus-ECL (Perkin Elmer, Inc., Waltham, MA, NEL104001EA), according to the manufacturer's recommendations.
4
Western Blot Analysis of Cellular Proteins
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Cultured cells were treated with the indicated conditions, and proteins were extracted with radio-immunoprecipitation assay buffer (#9806, Cell Signaling Technology Inc., Danvers, MA). Total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting. Western blotting was assessed with the ChemiDoc™ Imaging System (Bio-Rad Laboratories Inc.). The quantification of protein signals was performed with Image Lab software according to the manufacturer's protocol (Bio-Rad Laboratories Inc.). Primary antibodies and their dilutions were as follows: anti-TTF-1 (1 : 2000, Abcam; ab76013), anti-pro+mature SPB (1 : 5000, Abcam; ab40876), polyclonal antibody to SPC (1 : 300, Cloud-Clone Corp., Katy, TX, PAB623Mu02), and purified mouse anti-clathrin heavy chain (1 : 1000, BD Biosciences, Franklin Lakes, NJ, 610500). Secondary antibodies were anti-rabbit IgG and horseradish peroxidase- (HRP-) conjugated antibodies (1 : 2000, Cell Signaling Technology Inc., 7074) and anti-mouse IgG and HRP-conjugated antibodies (1 : 2000, Cell Signaling Technology Inc., 7076). The proteins were visualized with enhanced Western Lightning® Plus-ECL (Perkin Elmer Inc., Waltham, MA, NEL104001EA) according to the manufacturer's recommendations.
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