Human p53 was generated in-house for anti-p53 autoantibody detection experiments. Plasmid encoding human p53 (hp53; Addgene) was inserted into the pET28a bacterial expression vector. hp53 was first amplified by PCR with the following primer set: 5′-AAAGGATCCATGGAGGAGCCGCAGTCAGA-3′ and 5′-AAAGAATTCCAGGTGGCTGGAGTGAGCCC-3′. The PCR product was cloned into the Bam HI/Eco RI sites of the pET28a vector to create pET28a-hp53. This plasmid was transformed into Escherichia coli BL21 competent cells (Novagen). Protein expression was induced with 1 mM isopropyl-β-d -thiogalactopyranoside at 37°C for 5 hours. Bacteria were lysed, and the soluble fraction was collected. Recombinant protein was purified by affinity chromatography on Ni–nitrilotriacetic acid agarose (QIAGEN) as per the manufacturer’s instructions. Purified p53 was verified by 10 to 15% gradient SDS–polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad) and Coomassie brilliant blue (Thermo Fisher Scientific) staining, dialyzed with PBS, and stored at −80°C in PBS containing 20% glycerol.
Sds polyacrylamide gel electrophoresis sds page
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SDS–polyacrylamide gel electrophoresis (SDS-PAGE) is a laboratory technique used to separate proteins based on their molecular weight. It is a type of gel electrophoresis that uses sodium dodecyl sulfate (SDS) to denature and apply a negative charge to proteins, allowing them to be separated by size as they migrate through a polyacrylamide gel under the influence of an electric field.
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Lab products found in correlation
Bca assay, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (2 mentions)
Anti raga d8b5, by Cell Signaling Technology (2 mentions)
Anti aid maid 2, by Thermo Fisher Scientific (2 mentions)
Lamin b 66095 1 ig, by Proteintech (2 mentions)
Tfe3 hpa023881, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Phosphatase inhibitor cocktail, by Merck Group (2 mentions)
Anti tfeb a303 673a, by Fortis Life Sciences (2 mentions)
Anti raptor 24c12, by Cell Signaling Technology (2 mentions)
Tubulin 11224 1 ap, by Proteintech (2 mentions)
Coomassie brilliant blue, by Thermo Fisher Scientific (1 mentions)
Ni nitrilotriacetic acid agarose, by Qiagen (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Epigallocatechin gallate (egcg), by Merck Group (1 mentions)
Phospho gsk3β, by Cell Signaling Technology (1 mentions)
Anti p21, by Abcam (1 mentions)
Interleukin 6 (il 6), by Cell Signaling Technology (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
6 protocols using sds polyacrylamide gel electrophoresis sds page
1
Production and Purification of Human p53
2
Biochemical Analyses of Cellular Responses
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Bovine serum albumin (BSA), sodium dodecyl-sulphate (SDS) and EGCG were obtained from Sigma-Aldrich Canada (Oakville, ON). Phosphate-buffered saline (PBS) buffer solution (pH ~ 7.4) was purchased from (Wisent, St-Bruno, QC). For the SDS–polyacrylamide gel electrophoresis (SDS-PAGE) as well as the enhanced chemiluminescence (ECL) reagents, they were from Bio-Rad (Mississauga, ON). Micro bicinchoninic acid protein assay reagents were purchased from Pierce (Rockford, IL). The antibodies against BIP, P16, IL-6, phospho-AKT, AKT, phospho-GSK3β, and GSK3β were obtained from Cell Signaling Technology Inc (Danvers, MA). The anti-Tubulin antibody was purchased from ICN Biomedical (Aurora, OH), anti-P21 was from Abcam (Cambridge, UK), and anti-CD9, CD63 and CD81 from ThermoFisher Scientific (Waltham, MA). Horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA).
3
Immunoblotting of Submandibular Gland and Saliva
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Immunoblotting was performed as described previously [6 (link)] using submandibular gland tissue homogenates and saliva. Briefly, submandibular glands were homogenized in a radioimmunoprecipitation assay lysis buffer and were quantified for total protein using a Bio Rad Protein assay buffer (Bio-Rad, Hercules, CA, USA). About 30 µg of total protein extract was separated on SDS-polyacrylamide gel electrophoresis (SDS-PAGE; BioRad, Hercules, CA, USA) and transferred to a polyvinylidene fluoride membrane (PVDF). Later, membranes were probed with indicated primary antibodies and incubated again with the secondary antibody. Post incubation blots were developed using a chemiluminescence detection system. For Coomassie Brilliant Blue R-250 staining (CBB), samples were stained with CBB and de-stained using a 30% methanol/10% acetic acid solution.
4
SDS-PAGE Immunoblotting Reagents
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Reagents for SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were from Bio-Rad Laboratories (Hercules, CA). Tris, aprotinin, adenosine triphosphate (ATP), dithiothreitol, phenylmethylsulfonyl fluoride, Triton X-100, Tween 20, glycerol, and bovine serum albumin (BSA) (fraction V) were from Sigma Chemical Co. (St. Louis, MO). Nitrocellulose paper (BA85, 0.2 mm) was from Schleicher & Schuell (Keene, NH). Ketamine hydrochloride was from Cristália (Itapira SP, Brazil). The chemiluminescent kit was from Thermo Fisher Scientific (Rockford, IL, USA). The following antibodies were used: Anti-IL6 (M-19) and anti–α-tubulin (B-7) were from Santa Cruz Biotechnology; anti-pERK1/2 (Thr202/Tyr204), anti-p44/42 MAPK (ERK1/2), anti-pACC (Ser79), anti-ACC, anti–pAMPK (Thr172), anti-AMPKα, and anti–α-tubulin were from Cell Signaling Technology. Secondary antibodies were from Thermo Fisher Scientific. Recombinant IL6 was from Calbiochem (San Diego, CA, USA). PD98059 was from LC Laboratory (Woburn, MA). Unless otherwise specified, routine reagents were purchased from Sigma Chemical Co. (St. Louis, MO). The doses administered in each experimental group are given below.
5
Quantitative Western Blot Analysis of B Cell Signaling
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B cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich). Protein concentration was detected by BCA assay (Thermo Fisher Scientific), and an equal amount of protein was resolved in 4–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad). Proteins were transferred to polyvinylidene difluoride membranes (Millipore) and probed overnight with the following primary antibodies: : anti-p-S6K (108D2), anti-p-S6 (D57.2.2E), anti-LAMP1(C54H11), anti-p-4EBP1 (236B4), anti-Raptor (24C12), and anti-RagA (D8B5) all from Cell Signaling Technology), anti-AID (mAID-2, Thermo Fisher Scientific), anti-TFEB (A303–673A, Bethyl Laboratories), Lamin B (66095–1-Ig, Proteintech), tubulin (11224–1-AP, Proteintech), TFE3 (HPA023881, Sigma-Aldrich) and anti-β-actin (13E5, Sigma-Aldrich). The membrane was washed and incubated with indicated secondary antibody for the subsequently enhanced chemiluminescence (ECL, Thermo Fisher) exposure.
6
Western Blot Analysis of Signaling Pathways
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B cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) supplemented with protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich). Protein concentration was detected by BCA assay (Thermo Fisher Scientific), and an equal amount of protein was resolved in 4–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad). Proteins were transferred to polyvinylidene difluoride membranes (Millipore) and probed overnight with the following primary antibodies: anti-p-S6K (108D2), anti-p-S6 (D57.2.2E), anti-LAMP1(C54H11), anti-p-4EBP1 (236B4), anti-Raptor (24C12), and anti-RagA (D8B5) all from Cell Signaling Technology), anti-AID (mAID-2, Thermo Fisher Scientific), anti-TFEB (A303–673A, Bethyl Laboratories), Lamin B (66095–1-Ig, Proteintech), tubulin (11224–1-AP, Proteintech), TFE3 (HPA023881, Sigma-Aldrich) and anti-b-actin (13E5, Sigma-Aldrich). The membrane was washed and incubated with indicated secondary antibody for the subsequently enhanced chemiluminescence (ECL, Thermo Fisher) exposure.
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