WJMSCs and sEVs were lysed with RIPA buffer containing a Halt protease Inhibitor single‐use cocktail (Thermo Scientific). Membrane‐bound PD‐L1 were extracted using Mem‐PER™ plus membrane protein extraction kit following the manufacturer's instruction (Thermoscientific, USA). Protein lysates were separated by a Mini‐protean TGX precast gel (BIO‐RAD, USA) and transferred onto a PVDF membrane (BIO‐RAD, USA). Western blots were performed according to the standard techniques. The following antibodies were used at a dilution of 1:1000 in 5% nonfat milk unless otherwise stated: goat anti‐human PD‐L1 and PDL2 (R&D), Rabbit anti‐human CD90, CD105 (Thermo Scientific), Rabbit anti‐human CD9, CD81, HSP70, and CD63 (System Biosciences) and mouse anti‐human β‐actin (1:10,000, Sigma). Secondary antibodies include donkey anti‐goat‐HRP (R&D), goat‐anti‐rabbit‐HRP (Cell Signalling) and goat anti‐mouse antibody conjugated with HRP (Sigma). Signals were developed by using Pierce ECL Western Blotting Substrate (Thermo Scientific).
Donkey anti goat hrp
Manufactured by R&D Systems
Sourced in United States
Donkey anti-goat HRP is a lab equipment product that is used for the detection and quantification of goat-derived proteins in various biological samples. It is a conjugate of a donkey-derived antibody and the enzyme horseradish peroxidase (HRP). This product is commonly used in immunoassays, Western blotting, and other applications that require the specific detection of goat-derived proteins.
Automatically generated - may contain errors
Lab products found in correlation
Sample loading buffer, by Thermo Fisher Scientific (2 mentions)
Sample reducing agent, by Thermo Fisher Scientific (2 mentions)
Streptavidin horseradish peroxidase hrp, by Jackson ImmunoResearch (2 mentions)
Donkey anti goat hrp, by Jackson ImmunoResearch (2 mentions)
Acrylamide gradient, by Thermo Fisher Scientific (2 mentions)
Iblot2 apparatus, by Thermo Fisher Scientific (2 mentions)
Anti human igg biotinylatedantibody, by Southern Biotech (2 mentions)
Unlabeled goatanti human igm, by Southern Biotech (2 mentions)
Alas4000 imager, by GE Healthcare (2 mentions)
Mouse anti human β actin, by Merck Group (1 mentions)
Halt protease inhibitor single use cocktail, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit hrp, by Cell Signaling Technology (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Mem per plus membrane protein extraction kit, by Thermo Fisher Scientific (1 mentions)
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Santa Cruz Biotechnology (1 mentions)
Polyacrylamide gel, by Bio-Rad (1 mentions)
0.45 m pvdf membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
4 protocols using donkey anti goat hrp
1
Membrane Protein Extraction and Characterization
2
Antibody Isotype Detection by Western Blot
3
Antibody Isotype Detection by Western Blot
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B cell supernatants containing secreted antibodies were diluted in
H2O, 4x sample loading buffer (Life Technologies) and 10x sample reducing agent
(Life Technologies) and loaded onto precast gels with a 4-12% acrylamide gradient
(Invitrogen). The iBlot2 apparatus (Life Technologies) was used for protein transfer to
PVDF membranes followed by blocking for 1 h at room temperature with 3% BSA in TBS. The
membrane was incubated with different combinations of primary and secondary antibodies
diluted in TBS/1% BSA for 1 h at room temperature with 2 sequential TBS incubations to
wash the membrane between incubations. For detection of IgG, anti-human IgG-biotinylated
antibody (Southern Biotech, 2040-08) was used at 1 µg ml-1, followed by
25 ng ml-1 streptavidin-horseradish peroxidase (HRP) (Jackson ImmunoResearch,
016-030-084). IgM isotypes were stained with 10 µg ml-1 unlabeled goat
anti-human IgM (Southern Biotech, 2020-01) and 8 ng ml-1 donkey anti-goat HRP
(Jackson ImmunoResearch, 705-036-147). To detect LAIR1-containing antibodies, a polyclonal
goat anti-human LAIR1 antibody (R&D) at 2 µg ml-1 was combined
with secondary donkey anti-goat HRP. Membranes were developed with ECL-substrate on a
Las4000 imager (General Electric Company).
H2O, 4x sample loading buffer (Life Technologies) and 10x sample reducing agent
(Life Technologies) and loaded onto precast gels with a 4-12% acrylamide gradient
(Invitrogen). The iBlot2 apparatus (Life Technologies) was used for protein transfer to
PVDF membranes followed by blocking for 1 h at room temperature with 3% BSA in TBS. The
membrane was incubated with different combinations of primary and secondary antibodies
diluted in TBS/1% BSA for 1 h at room temperature with 2 sequential TBS incubations to
wash the membrane between incubations. For detection of IgG, anti-human IgG-biotinylated
antibody (Southern Biotech, 2040-08) was used at 1 µg ml-1, followed by
25 ng ml-1 streptavidin-horseradish peroxidase (HRP) (Jackson ImmunoResearch,
016-030-084). IgM isotypes were stained with 10 µg ml-1 unlabeled goat
anti-human IgM (Southern Biotech, 2020-01) and 8 ng ml-1 donkey anti-goat HRP
(Jackson ImmunoResearch, 705-036-147). To detect LAIR1-containing antibodies, a polyclonal
goat anti-human LAIR1 antibody (R&D) at 2 µg ml-1 was combined
with secondary donkey anti-goat HRP. Membranes were developed with ECL-substrate on a
Las4000 imager (General Electric Company).
4
Immunoblotting of UCP2 in Cells
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LCLs were lysed using RIPA lysis buffer containing 1% NP40, 0.1% SDS, 1% PMSF, 1% protease inhibitor cocktail and 1% sodium orthovanadate (Santa Cruz, Dallas, TX, USA). Protein concentration was determined using a BCA Protein Assay Kit (BioRad, Hercules, CA, USA), and lysates were prepared with 4X Laemmli Sample Buffer (BioRad) and 5% beta-mercaptoethanol. Samples were boiled for 5 min and cooled on ice for 5 min, and 50 µg of protein per lane was electrophoresed on a 10% polyacrylamide gel (BioRad) and transferred to a 0.45 µM PVDF membrane (Millipore, Billerica, MA, USA). Transfer efficiency was tested by Ponceau S staining (Santa Cruz) of gels. Membranes were probed overnight at 4°C with goat anti-UCP2 (1 µg/ml, R&D Systems, Minneapolis, MN, USA) after blocking with 2% non-fat milk. For detection, the membranes were incubated with donkey anti-goat-HRP (1∶5000, R&D Systems) and the blots were visualized using enhanced chemiluminescence (Thermo Scientific, Pittsburgh, PA, USA) and quantitated using ImageJ (National Institutes of Health, Bethesda, MD, USA). The membrane was stained for total proteins using the Reversible Protein Stain Kit (Pierce, Inc) and the darkest band was quantitated using ImageJ.
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