Proteins from lysates separated by SDS-PAGE were transferred to PVDF membrane (Hybond-P) at 30 V for 1 h. The membrane was then blocked overnight at 4 °C in 5% dried nonfat milk in Tris-buffered saline with 0.1% Tween 20 (v/v, TBST) or 0.5% BSA (w/v)/0.02% (w/v) SDS in phosphate-buffered saline with 0.1% Tween 20 (v/v, PBST). After blocking, the membranes were washed in 1X TBST or 1X PBST and incubated with primary antibodies (1:10000 rabbit anti-EF1A, Kerafast, ED7001) diluted into 1% dried nonfat milk in 1X TBST or 0.5% BSA/ 0.20% SDS in PBST, as indicated, for 1.5 h at 4 °C. After washing with the respective buffers, the membrane was incubated with anti-rabbit IgG-HRP (1:6666; Cell Signaling, 7074) secondary antibody in 1% dried nonfat milk or LICOR anti-goat fluorescent antibody in 0.5% BSA/ 0.02% SDS in PBST for 1 h at room temperature. ECL was used to visualize bands probed with HRP secondary antibody (Amersham Biosciences ECL Prime Western blotting, GE Healthcare, RPN2232) and LICOR Odyssey imager for the fluorescent probe. After probing, membranes were stained with Ponceau S or Coomassie to determine transfer efficiency.
Ecl prime western blotting
Manufactured by GE Healthcare
ECL Prime Western blotting is a sensitive chemiluminescent detection system for Western blot analysis. It is used to detect and quantify specific proteins in complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit igg hrp, by Cell Signaling Technology (1 mentions)
Nuclear extraction kit, by Abcam (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Molecular imager chemidoc xrs system, by Bio-Rad (1 mentions)
Anti flag, by Merck Group (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
3 protocols using ecl prime western blotting
1
Western Blot Protein Detection Protocol
2
Western Blot Analysis of NF-κB Localization
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Cells were harvested and lyzed using RIPA lysis and extraction buffer (Thermo Scientific) following manufacturer's protocol. Twenty to 30 μg of total protein lysates was loaded on Mini-PROTEAN TGX precast gels (Bio-Rad). Separated proteins were transferred to a polyvinylidene difluoride (PVDF) membrane using Trans-Blot Turbo Transfer System (Bio-Rad) followed by incubation with primary and horseradish peroxidase (HRP)–conjugated secondary antibodies [anti-mouse, catalog no. 7076S, Cell Signaling Technology (CST) or anti-rabbit, catalog no. 7074S, CST] and ECL Prime Western blotting (GE Healthcare) detection. For fractionation, cells were grown in 10-cm tissue culture plates and treated with 30 or 60 μmol/L of 3′-dA or NUC-7738 for 6 hours. Cells were grown in 10-cm tissue culture plates and fractionated using the Nuclear Extraction Kit (Abcam) following manufacturer's instructions. Twenty-five micrograms of each fraction was used for Western blot (WB) detection. NF-κB p65 antibody (catalog no. 8242, CST), nuclear marker Lamin B1 (catalog no. ab16048, Abcam), cytosolic GAPDH (catalog no. 97166, CST), cleaved PARP (catalog no. 9541, CST), and Actin (MAB1501, Merck) were used.
3
Western Blot Analysis of H-NS Proteins
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Whole-cell protein extracts (pelleted cells resuspended in Laemmli sample buffer) were separated on a 15% SDS-polyacrylamide gel. To detect specific proteins, they were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) by semidry electrotransfer. Prior to the Western blotting procedure, the total protein content of whole-cell extracts was checked by Coomassie blue staining.
For immunodetection of H-NS2-FLAG protein, a primary monoclonal anti-FLAG (Sigma) was used. To immunodetect H-NS native protein, a primary polyclonal anti-H-NS was used. Detection of primary antibodies bound to the specific proteins analyzed was performed with a horseradish peroxidase (HRP)-conjugated secondary antibody. The detection reagent used was ECL Prime Western blotting (GE Healthcare). Detection and the visualization of the chemiluminescent bands corresponding to the proteins being studied were performed using Molecular Imager ChemiDoc XRS system and Quantity One software (Bio-Rad).
For immunodetection of H-NS2-FLAG protein, a primary monoclonal anti-FLAG (Sigma) was used. To immunodetect H-NS native protein, a primary polyclonal anti-H-NS was used. Detection of primary antibodies bound to the specific proteins analyzed was performed with a horseradish peroxidase (HRP)-conjugated secondary antibody. The detection reagent used was ECL Prime Western blotting (GE Healthcare). Detection and the visualization of the chemiluminescent bands corresponding to the proteins being studied were performed using Molecular Imager ChemiDoc XRS system and Quantity One software (Bio-Rad).
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