Total lysates from human SCs were prepared and denatured as described previously [23 (link)]. Equal protein samples (usually 1–5 μg protein/lane) were subjected to polyacrylamide gel electrophoresis under denaturing conditions and then transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA) by a liquid transfer system (BioRad). Membranes were blocked with ECL blocking agent (GE Healthcare) in Tris-Buffer saline containing 0.05% Tween-20 (TBS-T) and incubated overnight with a 1:500–1:2000 dilution of each primary antibody or undiluted hybridoma culture supernatant. The membranes were washed with TBS-T prior to incubation with HRP-conjugated secondary antibodies. Immunoreactive protein bands were detected by enhanced chemiluminescence using ECL Advanced or Plus (GE Healthcare) based on signal intensity. Western blot films (Hyperfilm ECL) were scanned at a resolution of 400 dpi and quantified using Image Studio Lite Software (version 5.2). The expression of β-actin served as a control for the equal loading of protein samples.
Liquid transfer system
Manufactured by Bio-Rad
Sourced in United States
The Liquid Transfer System is a precision laboratory instrument designed to accurately and efficiently transfer liquids. It facilitates the movement of various solutions, reagents, and samples between containers, enabling researchers to carry out their experiments with consistency and reliability.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ecl blocking agent, by GE Healthcare (1 mentions)
Skim milk, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by New England Biolabs (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Diaminobenzidine dab solution, by Roche (1 mentions)
Bis tris gel, by Thermo Fisher Scientific (1 mentions)
Chemidoc chemoluminescence detection system, by Bio-Rad (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Bradford assay, by Bio-Rad (1 mentions)
Phosphatase inhibitor, by Roche (1 mentions)
Anti rabbit 7074, by Cell Signaling Technology (1 mentions)
Darq 7 camera, by Vilber (1 mentions)
Fusion solo 4s system, by Vilber (1 mentions)
Mini protean 3 system, by Bio-Rad (1 mentions)
6 protocols using liquid transfer system
1
Western Blot Analysis of Human SCs
2
Recombinant E. coli Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant E. coli Top10 cells were grown in LB medium containing ampicillin (100 µg/ml) at 37°C overnight. In the next day, 500 ml LB medium was inoculated with 5 ml Top10 cells cultured overnight. The inoculated cultures were grown with agitation under aerobic conditions at 37°C to reach OD600nm~0.5. Then the expression of the cloned genes was induced by different concentrations of arabinose (final concentration 0.002-20%). After incubation for 4 h, the cells were harvested by centrifugation at 4°C for 15 min and stored at -20°C until further use.
To analyze the expression of proteins by sodium SDS-PAGE, the bacterial pellets were suspended in a loading buffer and heated at 95°C for 5 min. Then 30 µl of each sample was subjected to 12-15% SDS-PAGE gels. For Western-blot analysis, the samples were separated by SDS-PAGE and transferred to nitrocellulose membrane (Schleicher and Schuell, Germany) using a liquid transfer system (Bio-Rad, USA). Membranes were blocked with skim milk (Merck, Germany) in PBST (PBS 1%+Tween20) and then washed with PBST. The membranes were incubated with the horseradish peroxidase-conjugated His-tag antibody (Invitrogen, USA), and the positive signals were detected by DAB (3,3′-diaminobenzidine) (Invitrogen, USA) and H2O2 as substrate.
To analyze the expression of proteins by sodium SDS-PAGE, the bacterial pellets were suspended in a loading buffer and heated at 95°C for 5 min. Then 30 µl of each sample was subjected to 12-15% SDS-PAGE gels. For Western-blot analysis, the samples were separated by SDS-PAGE and transferred to nitrocellulose membrane (Schleicher and Schuell, Germany) using a liquid transfer system (Bio-Rad, USA). Membranes were blocked with skim milk (Merck, Germany) in PBST (PBS 1%+Tween20) and then washed with PBST. The membranes were incubated with the horseradish peroxidase-conjugated His-tag antibody (Invitrogen, USA), and the positive signals were detected by DAB (3,3′-diaminobenzidine) (Invitrogen, USA) and H2O2 as substrate.
3
Western Blot Protocol for Cell Lysate Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed as previously described79 (link) with minor modifications. Briefly, 1 × 107 cells were lysed in the lysis buffer. Whole-cell lysates were fractionated by SDS-PAGE and the proteins were transferred to a PVDF membrane using a liquid transfer system (Bio-Rad). The membrane was blocked in 10% non-fat milk in TBST (1 × TBS with 0.1% Tween-20) for 1 h at room temperature followed by incubation with a primary antibody in 5% BSA (NEB) at 4 °C overnight with continuously shaking. After repeated washing, the membrane was then incubated in 10% non-fat milk containing a horseradish peroxidase conjugated secondary antibody for 1 h and washed in 1× TBST. The resultant bands were visualized by Clarity™ Western ECL Substrate (Bio-Rad). Primary antibodies and the dilution factors used are listed in Table S2 .
4
SDS-PAGE and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of the proteins was analyzed by SDS-PAGE. The bacterial pellets were suspended in a loading buffer, heated for 5 minutes at 95°C, and 25 µL of each sample was subjected to 15% SDS-PAGE gel. For the western blot, the samples were separated by the SDS-PAGE and transferred in nitrocellulose membranes by using a liquid transfer system (Bio-Rad). The membranes were blocked with skimmed milk in phosphate buffered saline (PBST) (PBS 1% + Tween 20) and then washed several times with PBST. The membranes were incubated with the conjugated His-tag antibody (Roche) for 1 hour at room temperature and developed by Diaminobenzidine (DAB) solution (Roche, Germany).
5
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Confluent cells were lysed in 100 mM Tris pH 7.5, 150 mM NaCl, 0.5% NP40, 0.5% triton-X100, 10% glycerol,1X protease inhibitor cocktail (Roche) and 1 X phosphatase inhibitor (Roche) for 20 min at 4 °C. After 15 min centrifugation at 13,000 g, solubilized proteins were recovered in the supernatant. Protein concentration was measured using Bradford assay (Bio-Rad). For SDS PAGE, 50 µg protein extracts were loaded in 4–12% Bis-Tris gel (Invitrogen) or poly-acrylamide gels and proteins were transferred overnight at 4 °C on a nitrocellulose membrane using a liquid transfer system (Bio-Rad). Non-specific sites were blocked with 5% non-fat dry milk in PBS 0.1% Tween 20. Primary antibodies were diluted (1/1000 to 1/500) in PBS 0.1% Tween 20 and incubated overnight at 4 °C. After three washes in PBS 0.1% Tween 20, secondary HRP antibodies diluted in PBS 0.1% Tween 20 (1/10,000) were incubated for 1 hr and washed three times with PBS 0.1% Tween 20. Immunocomplexes of interest were detected using Supersignal west femto maximum sensitivity substrate (Thermo Fisher Scientific) and visualized with ChemiDoc chemoluminescence detection system (Bio-Rad). Quantification of Western blots by densitometry was performed using the Gel analyzer plug in from Image J.
6
Western Blot of Sarkosyl-Insoluble Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins in Sarkosyl-insoluble materials were mixed with either Laemmli buffer or native sample buffer (SDS and b-mercaptoethanol were omitted from Laemmli). For denaturating condition, samples were heated in Laemmli buffer at 100 C for 5 minutes and applied to 10% Tris-glycine SDS-PAGE using mini PROTEAN III system, according to the manufacturer's instructions (BioRad). For native PAGE, samples were applied with or without heating at 100 C for 5 minutes to 8% native gel [273 mmol/L Tris-HCl, pH 8.8, 8% acrylamide/bis-acrylamide (19:1), 0.1% ammonium persulfate, 0.1% TEMED] in tris-glycine running buffer (without SDS), and were transferred to nitrocellulose membrane (sc-3718, SCBT) using a liquid transfer system (BioRad, Hercules, CA). For Western blotting, the nitrocellulose membranes were blocked with 10% w/v semifat dry milk in tris-buffered saline (0.01 mol/L Tris, 0.15 mol/L NaCl, pH 7.4) and incubated with primary antibody for overnight. After several rinses, the membranes were incubated with antirabbit (7074; Cell Signaling, Leiden, the Netherlands) or anti-mouse (A6782; Sigma, St. Louis, MO) secondary antibodies conjugated with horseradish peroxidase and developed with the ECL system (Pico; ThermoFisher Scientific, Gent, Belgium). The chemiluminescent signal was captured using a Fusion SOLO 4S system (Vilber Lourmat) equipped with a DARQ-7 camera.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!