The phagolysosomes containing latex beads and the ECVs were resuspended in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer, and then separated by SDS-PAGE (4–12%, Bio-Rad, Hercules, CA, USA). After Coomassie blue staining, gel slices were digested with trypsin, and the resulting peptides were analyzed by nano-high-performance liquid chromatography tandem mass spectrometry (nano-HPLC-MS/MS) in the Protein Core Facility at the University of Texas Medical Branch (Galveston, TX, USA). LC-MS/MS results were searched against data in the International Protein Index (IPI) canine database using the Mascot search algorithm (v2.1, Matrix Science, London, UK). The search parameters were described below. Oxidation was used as variable modification; mass tolerance for peptide was set as 0.01 Da; and mass tolerance for fragment was set as 0.3 Da. The use of trypsin was specified and one missed cleavage was allowed. A cutoff expectation value of 0.01 (significance threshold) was used for MS/MS spectra that resulted in a false discovery rate of 2.40% (automatic decoy database search). Criteria of protein identification were as follows: minimum of two observed significant peptides; single peptide with E values of <0.005.
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SDS-PAGE is a technique used to separate proteins based on their molecular weight. It involves the use of sodium dodecyl sulfate (SDS) to denature the proteins and an electric field to drive the proteins through a polyacrylamide gel. The distance the proteins travel through the gel is inversely proportional to their molecular weight, allowing for separation and identification.
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Lab products found in correlation
Pvdf membrane, by Merck Group (119 mentions)
Pvdf membrane, by Bio-Rad (48 mentions)
Nitrocellulose membrane, by Bio-Rad (29 mentions)
Protease inhibitor cocktail, by Roche (26 mentions)
Polyvinylidene difluoride membrane, by Merck Group (26 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (25 mentions)
Ripa lysis buffer, by Beyotime (25 mentions)
Ripa buffer, by Merck Group (24 mentions)
Chemidoc xrs system, by Bio-Rad (24 mentions)
Ripa buffer, by Beyotime (22 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (19 mentions)
Chemidoc mp imaging system, by Bio-Rad (18 mentions)
Ripa buffer, by Thermo Fisher Scientific (18 mentions)
Trans blot turbo transfer system, by Bio-Rad (16 mentions)
Bca protein assay kit, by Beyotime (16 mentions)
Protease inhibitor cocktail, by Merck Group (16 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (15 mentions)
Chemidoc xr, by Bio-Rad (15 mentions)
Image lab software, by Bio-Rad (14 mentions)
Polyvinylidene fluoride membrane, by Merck Group (14 mentions)
902 protocols using sds page
1
Proteomic Analysis of Phagolysosomes and ECVs
2
Quantifying Secreted MMP-1 and HIF Proteins
3
Co-immunoprecipitation and In Vivo Ubiquitination
4
Phospho-Zap-70 Immunoblotting Protocol
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About 1 × 107 CD4+ T-cells were stimulated with aCD3-/CD28-coated microbeads for 30 min in the presence/absence of PRBCs or 1 mM NAC. Thereafter, cells were washed thoroughly using RBC lysis buffer (154 mM NH4Cl, 10 mM KHCO3, and 100 μM Na2 EDTA). Washed CD4+ T-cells were lysed with radioimmunoprecipitation assay buffer supplemented with protease inhibitor and phosphatase inhibitors (all Sigma–Aldrich). After 30 min of incubation on ice with periodic pulse vortexing, cell lysates were centrifuged at 16,000g for 15 min at +4 °C. For equal loading, protein concentration was measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to manufacturing instructions. A 4%–12% SDS-PAGE (BioRad) was used to separate proteins following a transfer onto polyvinylidene difluoride membranes (GE Healthcare). The membrane was blocked with bovine serum albumin (Sigma–Aldrich), and the following antibodies were used for incubation overnight: Phospho-Zap-70 (Tyr319)/Syk (Tyr352) (1:2000, #2701; Cell Signaling Technology) and total Zap-70 (1:1000, 99F2; Cell Signaling Technology). Protein bands were visualized using horseradish peroxidase–linked anti-rabbit (Cell Signaling Technology) or antigoat (Abcam) antibodies and SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).
5
Osteoclast Signaling Pathway Analysis
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Total lysates of human osteoclast precursor cells were prepared from the lysis buffer (Cell Signaling Technology). Total lysates were loaded on 4–12% SDS-PAGE (Bio-Rad), and then western blot analysis was performed using antibodies, pBtk Y223 (Novus Biologicals), pLyn Y396 (Gene Tex), Btk, Lyn, pGab2 Y452, Gab2, pPLCγ2 Y759, PLCγ2, pBLNK, BLNK, NFATc1 (Cell Signaling Technology).
6
Western Blot Analysis of Stress Response
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OPCs were washed twice with PBS and lysed in ice-cold RIPA buffer (Sigma) containing Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, PI78441). Lysates were then centrifuged at 12,000 rpm for 20 min at 4°C. A total 20 µg of protein lysates was separated by 4–12% SDS-PAGE (Bio-Rad, 4561095) and transferred to a nitrocellulose membrane. The following primary antibodies were used: anti-p-eIF2α (Abcam, ab32157, 1:2000), anti-T-eIF2α (Cell Signaling, 9722s, 1:1000), anti-puromycin (Millipore, MABE343, 1:2000), anti-BIP (Cell Signaling, 3177s, 1:1000), anti-GADD34 (Proteintech, 10449–1-AP, 1:500), anti-ATF4 (Santa Cruz, sc-390063, 1:500), anti-CHOP (Thermo Fisher, MAI-250, 1:500), anti-XBP-1-spliced (Cell Signaling, 82914s, 1:1000), and anti-actin (Sigma, A2066, 1:2000). Quantification of Western blot bands were performed by Image Lab Software (Bio-Rad).
7
Western Blotting Analysis of Frozen Cortex
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Human frozen cortex (350 mg) were homogenized on ice in anti-proteases/anti-phosphatases (Complete/ PhosSTOP, Roche) containing RIPA lysis buffer. Protein quantitation was assessed by BCA (Pierce) and samples (90 μg/lane) were resolved by 4-12% SDS-PAGE (Bio-Rad), electrotransferred to nitrocellulose membranes (Trans-Blot Turbo, Bio-Rad), followed by 5% BSA-blocking and incubation with primary antibodies (Supplementary Table 4 ) at 4°C overnight. After washing, membranes were incubated 1 hr at RT with the corresponding horseradish peroxidase-coupled secondary antibodies and visualized using the Pierce ECL Western Blotting Substrate (Thermo Scientific). Immunoblots images were captured using LAS4000 (GE Healthcare) and quantitation of the bands was performed by Image J free software (NIH).
8
Western Blot Analysis of Phospho-Smad3 and LC3
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Cells were lysed with RIPA buffer supplemented with protease inhibitor and phosphatase inhibitors (all Sigma Aldrich). After 30 minutes incubation on ice with periodic pulse vortexing, cell‐lysates were centrifuged at 16 000 g for 15 minutes at +4°C. For an equal loading, protein concentration was measured using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to manufacturing instructions. A 4%‐12% SDS‐PAGE (Bio‐Rad, Hercules, CA, USA) was used to separate proteins following a transfer onto PVDF membranes (GE Healthcare). The membrane was blocked with bovine serum albumin; BSA (Sigma Aldrich) and the following antibodies were used for incubation over night: phospho‐Smad3 (1:2000, Anti‐Smad3 PhosphoS423+S425, Abcam, Cambridge, UK), total Smad2/3 (1:1000 R&D Systems), LC3 A/B (1:1000 D3U4C, Cell Signaling Technology, Massachusetts, USA), and Pan‐Actin (1:2000, D18C11, Cell Signaling Technology). Protein bands were visualized using HRP‐linked anti‐rabbit (Cell Signaling Technology) or anti‐goat (Abcam) and SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific).
9
Protein Purity and Oligomeric State Analysis
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The purity and molecular weight of the proteins was assessed by loading 4 µg of denatured protein on a 4-12% SDS-PAGE (Biorad) under reducing conditions (100 mM dithiothreitol). Gels were stained in Coomassie brilliant blue R-250 for 1 hour and destained in 35% methanol and 10% acetic acid.
The oligomeric state of purified proteins was determined by size exclusion chromatography (SEC) using 50 µg of protein in a 300 μL loop connected to a Superose 6 Increase 10/300 GL (GE Healthcare Life Sciences) gel filtration column calibrated with a series of standards. Fractions of 1 mL were collected on the basis of elution volumes with peak absorbance values for subsequent analyses.
The oligomeric state of purified proteins was determined by size exclusion chromatography (SEC) using 50 µg of protein in a 300 μL loop connected to a Superose 6 Increase 10/300 GL (GE Healthcare Life Sciences) gel filtration column calibrated with a series of standards. Fractions of 1 mL were collected on the basis of elution volumes with peak absorbance values for subsequent analyses.
10
Protein Expression Analysis via Western Blot
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Cell lysates were run on a 4–12% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel (Bio-Rad, Hercules, CA, USA) and the proteins were transferred to a nitrocellulose membrane. The membrane was blocked with 5% milk in 1% TBST (Tween-20 tris-buffered saline) for 1 h at RT and then incubated with a primary antibody specific for iNOS (ab129372; Abcam) overnight at 4 °C. The membrane was washed and then incubated with the appropriate horseradish peroxidase secondary antibody for 1 h at RT. The protein blots were incubated with ECL substrates (Bio-Rad) for 5 min at RT and then detected on CL-Xposure Film (Thermo Scientific, Skokie, IL, USA) and developed.
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