The OB cells were seeded in gelatin-coated 6-well plates at a density of 200 × 103 cells per well in standard conditions for 96 h. The cells were treated with 150 μL of RIPA Buffer (Sigma Aldrich, St. Louis, MO, USA) and a protease inhibitor cocktail. The total protein level was measured by BSA assay kit (Thermo Scientific, Waltham, MA, USA) using Varioscan (Thermo Scientific, Waltham, MA, USA). An equal amount of protein from each donor was used for the assay. Extracts were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, CA, USA). Primary antibodies for RUNX2 were used for Westernblotting detection of RUNX2 protein.
Sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page
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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is a laboratory technique used to separate and analyze proteins based on their molecular weight. It involves the use of a polyacrylamide gel and an electric current to separate the proteins, which are denatured and coated with the anionic detergent sodium dodecyl sulfate (SDS) prior to the separation process.
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Lab products found in correlation
Triton x 100, by Merck Group (2 mentions)
Bsa assay kit, by Thermo Fisher Scientific (1 mentions)
Varioscan, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Beyotime (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Mupid one, by Takara Bio (1 mentions)
Milli q water purification system, by Merck Group (1 mentions)
Dextran t500, by GE Healthcare (1 mentions)
Western blot reagents, by Bio-Rad (1 mentions)
Kd025, by Merck Group (1 mentions)
Ficoll, by GE Healthcare (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
P stat5, by Cell Signaling Technology (1 mentions)
Stat5, by Cell Signaling Technology (1 mentions)
Mmp 11, by Cell Signaling Technology (1 mentions)
Epidermal growth factor (egf), by Cell Signaling Technology (1 mentions)
18 protocols using sodium dodecyl sulfate polyacrylamide gel electrophoresis sds page
1
Quantifying RUNX2 Protein Expression
2
Protein Extraction and Western Blot Analysis
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Cells were harvested after being washed twice with cold PBS (4 °C). Protein lysates were prepared using RIPA lysis buffer (Beyotime, China) including protease inhibitor cocktail (EDTA-free, mini-tablet) (Cat. No. HY-K0011, MedChemExpress Co., Ltd), and the protein concentration was measured using the BCA Assay Kit (Beyotime Institute of Biotechnology). Extracted proteins (30 µg/10 µl/lane) from each group were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad Laboratories, Inc.). After blocking with 5% non-milk for 70 min at room temperature, the membranes were incubated with specific primary antibodies at 4 °C overnight. Then, after washing three times with Tris-buffered saline containing Tween-20 (TBST), the membranes were incubated with secondary antibodies for 70 min at room temperature. Proteins were visualized by chemiluminescence using enhanced chemiluminescent substrate (New Cell & Molecular Biotech Co., Ltd). Immunoreactive bands were examined using the ChemiDoc Imaging System (Bio-Rad Laboratories, Inc.). Autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad). The GAPDH antibody was used as a control.
3
Agarose Gel Electrophoresis Protocol
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The procedure was conducted by mixing 1% agarose gel with 1× TAE buffer (Bioneer, Daejeon, Republic of Korea), and 1× TAE buffer solution. The 50× TAE buffer (Bioneer, Republic of Korea) was diluted 1× with water produced using a Milli-Q water purification system (Millipore, Billerica, MA, USA). A 1 kb ladder from GeneDireX (Taiwan, China) was used. Electrophoresis was conducted at 100 V for 60 min (Mupid-One; Takara, Japan). The sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad Laboratories, Hercules, CA, USA) method was adopted, as described by Bakhsh, Lee, Bakry, Rathnayake, Son, Kim, Hwang and Joo [19 (link)].
4
Characterization of ROCK2 Signaling in Neutrophils
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Dextran T500 and Ficoll were purchased from GE Healthcare. Dulbecco’s phosphate-buffered saline (PBS), bovine serum albumin (BSA), Hanks’ balanced salt solution (HBSS), fMLF, PMA, ATP, luminol (5-amine-2,3-dihydro-1,4-phtalazinedione), Y27632, KD025, and all the other chemical products used were obtained from Sigma Aldrich. Human granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin-4 (IL-4) were obtained from PeproTech France. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and western blot reagents were purchased from Bio–Rad (Marnes-la-Coquette, France). ROCK2 cDNA and active ROCK2 protein were purchased from the University of Dundee, School of Life Sciences Medical Sciences Institute, Scotland. Antibodies against phospho-(RRXS/Thr)-PKA substrate (mab#9624) and ROCK2 (used for IP: D1B1#9029) were from Cell Signaling Technology. An anti-ROCK2 antibody (used for IP#20248-1-AP) was from Proteintech. Antibodies against ROCK2 (D11, sc-398519), ROCK1(G6, sc-17794), ICAM1 (G5, sc-8439), GADPH (6C5, sc-32233), p40phox (B-1, sc-48376), and gp91phox (54.1, sc-130543) and secondary antibodies were purchased from Santa Cruz Biotechnology. Rabbit polyclonal antibodies against p47phox, p22phox, and p67phox and phosphorylated p47phox-sites were generated by our lab as previously described (32 (link), 33 (link), 42 (link)).
5
Regulation of MMP-9 Expression
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EGF and its inhibitors (LY294002 and U0126) were obtained from Sigma-Aldrich Corp (St. Louis, MO). Quinazoline (QNZ) was purchased from Enzo Life International Inc. (Plymouth Meeting, PA). EGFR, STAT3, STAT5α, STAT5β, p65, and scramble small interfering (si) RNA were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Complementary DNA (cDNA) plasmids containing MMP-9 were purchased from Origene Technologies (Rockville, MD) and were subcloned to the pCMV6-XL vector. Antibodies against β-actin, MMP-9, MMP-11, EGF, EGFR, phosphorylated (p)-ERK1/2, ERK1/2, p-AKT, AKT, p-STAT3, STAT3, p-STAT5, and STAT5 were purchased from Cell Signaling Technology, Inc. (Danvers, MA). All reagents for quantitative reverse transcription polymerase chain reaction (qRT-PCR) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were purchased from Bio-Rad Laboratories (Hercules, CA, USA). All other reagents were obtained from Invitrogen (Carlsbad, CA), unless otherwise specified.
6
Western Blot Analysis of SAAL1, PD-L1, and GAPDH
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Cells were lysed using lysis buffer (Cat. 9803S; Cell Signaling Technology Inc., Danvers, MA, USA) supplemented with a protease inhibitor cocktail (Cat. 11697498001; Roche Diagnostics, GmbH) for 30 min at 4° C. Protein concentration was measured by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Inc.). A total of 30 μg of protein per lane were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred to PVDF membranes (Merck Life Sciences, Inc., Australia). After blocking for 2 h with 5% skimmed dried milk, the membranes were washed with 1×TBST (1×Tris-Buffered Saline, 0.1% Tween 20) and incubated at 4° C overnight with rabbit polyclonal primary antibodies (1:1000 dilution; Novus Biologicals Inc., UK) against SAAL1 (Cat. NBP1-83447), PD-L1 (Cat. NBP1-76769), and GAPDH (Cat. NB300-327). After 1×TBST wash, the membranes were incubated with an anti-rabbit IgG secondary antibody (Cat. NBP1-75293; 1:3000 dilution; Novus Biologicals, Inc.) at room temperature for 2 h. Immunoreactive bands were detected using an ECL western blotting system (Clarity Western ECL Substrate; Bio-Rad Laboratories, Inc.). Band densities were measured using ImageJ software, and normalized to those of GAPDH.
7
Stability Analysis of NDV_LS/L289A_S-F Virus
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The allantoic fluid containing the NDV_LS/L289A_S-F virus was harvested and clarified by centrifugation. The clarified allantoic fluid was aliquoted into 15 mL volumes. Week (wk) 0 allantoic fluid was concentrated immediately after centrifugation as described above, through a 20% sucrose cushion. The pelleted virus was re-suspended in 300 µL phosphate buffered saline (PBS) and stored at −80 °C. The other three aliquots of the allantoic fluid were maintained at 4 °C to test the stability of the S-F construct. Week 1, 2, and 3 samples were collected consecutively on a weekly basis, and concentrated virus was prepared in 300 µL PBS using the same method. The protein content of the concentrated virus from wk 0, 1, 2, and 3 was determined using the BCA assay after one freeze-thaw from −80 °C. One microgram of each concentrated virus preparation was resolved in 4–20% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; Bio-Rad, Hercules, CA, USA). The S-F protein and the NDV hemagglutinin-neuraminidase (HN) protein were detected by Western blot.
8
TPPU and L-NAME Protein Detection
9
MERS-CoV S Glycoprotein Expression
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An expression construct encoding amino acids 358-571 (VEQA…CPKL) of the MERS S glycoprotein and a carboxy terminal 6x HIS tag was synthesized by Twist Biosciences and cloned into the pTwist CMV BetaGlobin WPRE Neo expression vector. Plasmids were produced in NEB 5-alpha competent Escherichia coli (New England Biolabs, Ipswich, MA, USA) and transfected into Expi293 cells using the Expi293 Expression System Kit (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions. Seven days following transfection, cleared supernatants were passed over a HisTrap HP column (Cytiva), and bound protein was eluted with 450 mM imidazole. Buffer exchange to PBS and protein concentration was performed using centrifugal protein concentrators with a 10 kDa MW cutoff (Pierce, Appleton, WI, USA). Final protein concentration was determined by BCA protein assay (Pierce). MW and protein purity were confirmed by Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA) (Figure 2 ).
10
NIR Dye Labeling of Monoclonal Antibodies
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