Cell monolayers were incubated on ice with RIPA lysis buffer (Beyotime, P0013B) containing a protease inhibitor cocktail (Roche, 4693132001) and a phosphatase inhibitor cocktail (Roche, 4906845001). The lysates were then clarified by centrifugation at 16,000 × g for 5 min at 4°C, and the protein concentration was quantified using the BCA protein assay kit (Thermo Scientific, 23225). Equal amounts of protein samples were denatured for 5 min in 2 × SDS-PAGE loading buffer (Sigma-Aldrich, S3401). Proteins were separated on SDS-PAGE gels and then electrotransferred onto nitrocellulose membrane (Bio-Rad, 162-0115) or polyvinylidene fluoride membranes (Millipore, ISEQ00010), which were then blocked for 1 h at room temperature in Tris-buffered saline (Sigma-Aldrich, V900838-4L) containing 5% nonfat milk powder and 0.1% Tween 20 (Sigma-Aldrich, P7949). Next, the membranes were incubated with primary antibodies at 4°C overnight and then with the corresponding secondary antibodies conjugated to horseradish peroxidase at room temperature for 1 h. The protein bands were detected using the ECL kit (Thermo, 34080).
Sds page loading buffer
Manufactured by Merck Group
Sourced in United States
SDS-PAGE loading buffer is a solution used to prepare protein samples for separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The buffer contains SDS, a detergent that denatures proteins and gives them a uniform negative charge, allowing them to be separated based on their molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (1 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Ripa buffer, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Protein assay reagent, by Beckman Coulter (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Protein a g sepharose beads, by Santa Cruz Biotechnology (1 mentions)
Protein g agarose, by Merck Group (1 mentions)
Phosphosafe extraction reagent, by Merck Group (1 mentions)
Pureproteome protein a magnetic beads, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Odyssey clx imaging system, by LI COR (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Beyotime (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
8 protocols using sds page loading buffer
1
Protein Extraction and Western Blot Analysis
2
Immunoprecipitation and Western Blot Analysis of Viral Proteins
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Cells were lysed with RIPA buffer (Cell Signaling) supplemented with 0.1 M phenylmethyl sulfonyl fluoride (PMSF), protease inhibitor mixture, and phosphatase inhibitors 2 & 3 (Sigma)). Protein concentrations for western blotting and immunoprecipitation were determined using the Bio-Rad protein assay reagent and a Beckman Coulter spectrophotometer. Immunoprecipitation experiments were conducted as described previously [63 (link)]. Briefly, 500 μg of protein was used for each immunoprecipitation and precleared with 50 μl protein A/G sepharose beads (Santa Cruz) for 6 h. Antibody (2 μg) was incubated with 20 μl protein A/G sepharose beads for 6 h to overnight. The immune-complexes were washed 3 times with RIPA buffer before being resolved using 2× SDS-PAGE loading buffer (sigma) and separated on a 4–20% Tris-HCl gradient SDS-PAGE gel (BioRad). The signal was detected using the Odyssey Infrared Imaging System (Li-Cor Biosciences). The following antibodies were used: Actin (sc-1616), Zta (Argene 11–007), Rta (Argene 11–008), BMRF1 (EBV-018-48,180), BGLF4 (Argent AP8057b), VCA (Argene 11–019), LMP1 (BD 559898) and GAPDH (Cell Signaling 2118 L).
3
Immunoprecipitation of GRP78 Complexes
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HEK293 cells transfected with GRP78 expression vectors were collected and extracted using lysis buffer (150 mM NaCl, 50 mM Tris, 1% NP-40, 1× protease inhibitor, and 1 mM phenylmethylsulfonyl fluoride, pH 7.6). The cell extracts were mixed overnight at 4°C with 10 μg of monoclonal anti-HA antibody, 2 μg of HA-tagged rhLK8, and protein G-agarose (Sigma). The immunoadsorbents were recovered by centrifugation for 5 min at 700×g, washed three times, and centrifuged (5 min at 700 × g) in IP buffer (150 mM NaCl, 50 mM Tris, 0.1% NP-40, pH 7.6). The pellets were resuspended in 30 μL of SDS-PAGE loading buffer (Sigma) and analyzed by Western blotting.
4
MAGI2 Protein Immunoprecipitation and Detection
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Cells were lysed in PhosphoSafe Extraction Reagent (Sigma) containing protease inhibitor cocktail and cell debris removed by centrifugation. MAGI2 protein was immunoprecipitated from cell lysates using the anti-MAGI2 (C3; 4 µg) antibody. To ensure specificity, a rabbit IgG isotype (4 µg) was also included as well as an additional control without any antibody. The samples were incubated at 4 °C overnight with gentle shaking. Pureproteome protein A-magnetic beads (10 µl) (Merck-Millipore) was added to all samples and controls and incubated at 4 °C for 90 min60 . The tubes were then placed in a magnetic stand and the supernatant removed, washed five times with PBST (1 ml) and the samples denatured in SDS-PAGE loading buffer (70 µl) (Sigma). Samples were then separated by SDS-PAGE, transferred to nitrocellulose membranes and blocked. The membranes were probed with a mouse anti-PTEN antibody (217702), as well as a rabbit anti-MAGI2 antibody and then developed as described above.
5
Western Blot Analysis of Cultured Cells
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Cultured cells were washed with ice-cold PBS and lysed in RIPA buffer (Beyotime, China) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor cocktails (1:100, v/v, Beyotime, China). Protein solution or whole cell lysates were resolved in SDS-PAGE loading buffer (Sigma-Aldrich, USA), transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA), and probed with the primary and secondary antibodies. Western blotting images were photographed using an Odyssey CLx Imaging System (LI-COR, USA).
6
Western Blot Analysis of Protein Expression
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PAMs were lysed in 200 μL RIPA lysis buffer (89900, ThermoFisher Scientific, USA) with protease inhibitor cocktail (78429, ThermoFisher Scientific, USA) for 30 min on ice. After centrifugation, the lysates were denatured for 10 min in 2✕ SDS-PAGE loading buffer (S3401, Sigma Aldrich). Samples were separated by electrophoresis on SDS-PAGE gels and then transferred to transfer nitrocellulose membranes (ISEQ00010, Merck-Millipore, USA). Next, membranes were blocked with 5% skimmed milk for 1 h at room temperature. After that, the membranes were incubated with primary antibodies at 4 °C overnight, and then incubated with corresponding secondary antibodies conjugated to HRP at room temperature for 1 h. Finally, the relative expression levels of protein were detected using ECL reagents (35055, ThermoFisher Scientific) and were quantified by Quantity One software (Bio-Rad Laboratories, USA).
7
Deubiquitination Assay of Transcription Factor
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De-ubiquitination assay was carried out using the UbiCREST Deubiquitinase Enzyme Kit (Boston Biochem). TF was immunoprecipitated from MDA-MB-231 cell lysate as above but at a larger scale using 20 µg of FL295 antibody and 100 µl of Protein A-magnetic beads and the samples were not eluted. Instead after washes with PBST, the samples were washed once with the "DUB reaction buffer" (1 ml) supplied with the kit and resuspended in the reaction buffer (450 µl). The reactions were prepared on ice, in ten tubes containing the nine DUB enzyme (5 µl) plus one control reaction, and 45 µl of DUB reaction buffer added. The reactions were started by adding 50 µl of magnetic bead slurry, mixed and incubated at 37°C for 30 min. The samples were then placed in the magnetic stand and the supernatant transferred to a fresh tube containing 2 × SDS-PAGE loading buffer (Sigma). The samples were boiled and analysed using western blot by probing with the FK2 anti-ubiquitin antibody.
8
Sperm Protein Extraction Protocol
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After capacitation, all three sperm treatment groups (proteasomally inhibited, non-inhibited and vehicle control) were washed three times with TBS, at the same time with washed cauda epididymal and ejaculated spermatozoa (described above), and used for protein extractions.
Approximately 100 million spermatozoa were mixed with 100 µL of SDS-PAGE loading buffer (Laemmli 1970) supplemented with 50 mM dithiothreitol (DTT; Sigma-Aldrich) and protease and phosphatase inhibitors (Thermo Fisher Scientific). Spermatozoa were left to denature/reduce in loading buffer at 80°C, 10 min. Spermatozoa were spun off, and extracts were stored in -25°C for further analysis.
Approximately 100 million spermatozoa were mixed with 100 µL of SDS-PAGE loading buffer (Laemmli 1970) supplemented with 50 mM dithiothreitol (DTT; Sigma-Aldrich) and protease and phosphatase inhibitors (Thermo Fisher Scientific). Spermatozoa were left to denature/reduce in loading buffer at 80°C, 10 min. Spermatozoa were spun off, and extracts were stored in -25°C for further analysis.
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