The testicular tissues or human SSCs with the treatment of miRNA oligonucleotides/overexpression plasmid were lysed with RIPA buffer. The concentrations of proteins were measured by the BCA kit. Thirty micrograms of cell lysate from each cell sample were used for SDS-PAGE (Bio-Rad). Then, the proteins were transferred into PVDF membranes (Roche, Germany) and blocked with 5 % non-fat dry milk (Carnation, CA). Subsequently, the samples were incubated with primary antibodies against EZH2 (ab191250, Abcam), GDNF (ab176564, Abcam), DAZL (ab34139, Abcam), Caspase-3 (ab32042, Abcam), Bax (ab32503, Abcam), and Bcl-2 (ab32124, Abcam) overnight at 4°C. The nitrocellulose membrane was incubated for 2 h after adding appropriate secondary antibodies (HRP-conjugated goat anti-rabbit) (Abcam). Finally, the expression of proteins was evaluated using enhanced chemiluminescence.
Non fat dry milk
Manufactured by Roche
Sourced in Germany
Non-fat dry milk is a powdered form of milk that has been processed to remove the fat content. It serves as a versatile ingredient for various laboratory applications, providing a reliable source of protein, carbohydrates, and essential vitamins and minerals.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti rabbit, by Abcam (2 mentions)
Tripure isolation reagent, by Roche (2 mentions)
Ab32042, by Abcam (1 mentions)
Ab191250, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Ab34139, by Abcam (1 mentions)
Ab176564, by Abcam (1 mentions)
Ab32124, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Roche (1 mentions)
Sds polyacrylamide gel, by Bio-Rad (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Ecl western blotting substrate, by Bio-Rad (1 mentions)
Polyvinylidene difluoride pvdf membrane, by Thermo Fisher Scientific (1 mentions)
4 protocols using non fat dry milk
1
Quantitative Protein Expression Analysis of Human SSCs
2
Protein Extraction and Analysis of SSCs
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A TriPure Isolation Reagent (Roche, Germany) was used for extracting the total protein from SSCs. Then, electrophoresis was employed to separate 20 µg of the total protein for each sample. In the next step, the proteins were transferred into 10.5% and 12.5% gradient sodium dodecyl sulfate-polyacrylamide gel (BioRad Laboratories, Hercules, CA) and polyvinylidene difluoride membranes (Roche, Germany), and then were blocked with 5% non-fat dry milk (Carnation, CA). Subsequently, the primary antibodies against ID4 and c-kit (1:1000) were used for incubating specimens for 24 hours at 4°C, and then secondary antibodies and HRP were added. Finally, the rates of protein expression were evaluated with enhanced chemiluminescence.
3
Protein Expression Analysis of SSCs
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TriPure Isolation Reagent (Roche, Germany) was used to extract total protein from SSCs. Electrophoresis was done to separate 20 µg of the total protein for each sample. Then, the proteins were transferred into 10.5 and 12.5 % gradient sodium dodecyl sulfate (SDS)-polyacrylamide gel (BioRad Laboratories, Hercules, CA) and polyvinylidene difluoride (PVDF) membranes (Roche, Germany) and blocked with 5 % non‐fat dry milk (Carnation, CA). Subsequently, the samples were incubated with primary antibodies against GFRα1 (1:1000), PLZF (1:1000) and ID4 (1:1000) overnight at 4 °C. Nitrocellulose membrane was incubated for 2 h after adding appropriate secondary antibodies (HRP conjugated goat anti‐rabbit) (Abcam). Finally, the expression of the proteins was evaluated using enhanced chemiluminescence.
4
Western Blot Analysis of Cartilage Proteins
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For western blot, cell lysates were isolated from NP tissues with RIPA lysis buffer (10 mM of Tris, pH 7.4, 100 mM of NaCl, 1 mM of EDTA, 1 mM of EGTA, 1 mM of NaF, 20 mM of Na 4 P2O 7 , 2 mM of Na 3 VO 4 , 1% Triton X-100, 10% glycerol, 0.1% sodium dodecylsulphate (SDS), and 0.5% deoxycholate). Protein concentrations were estimated using Bradford protein assay (Bio-Rad, Hercules, USA). An equal amount of protein (40 µg) was resolved using 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane (Thermo Fisher Scientific). Non-specific binding sites were blocked with 5% nonfat dry milk (Roche, Basel, Switzerland) in Tris-buffered saline containing 0.1% Tween (TBST). The membrane was incubated with primary antibody against rabbit COL2A1 or aggrecan (Abcam, Cambridge, UK) in TBST at 4°C overnight on a shaker, and was then incubated with horseradish peroxidase (HRP)-conjugated secondary anti bodies for 1 h at room temperature. The immunoblots were visualized using Western ECL Blotting Substrates (Bio-Rad). The protein bands were analyzed for densitometry using ImageJ software (National Institutes of Health, Bethesda, USA).
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