For protein level analyses, proteins were extracted from germ cells using RIPA buffer (Santa Cruz) containing 1× protease inhibitor cocktail (Roche). Protein concentration was calculated using a BCA protein assay kit (Pierce). Lanes of 4–15% gradient SDS polyacrylamide gels (Bio-Rad) were loaded with 20 µl of 1 mg/ml protein extract. Following protein separation via standard SDS PAGE, proteins were transferred to PVDF membranes using the Trans-Blot® Turbo™ western transfer system (Bio-Rad). Primary antibodies and dilution used are presented in Supplemental Table S2 . At a 1∶20,000 dilution, Invitrogen horseradish peroxidase-conjugated antibodies rabbit anti-mouse (R21455), goat anti-rabbit (A10533), rabbit anti-goat (R21459) were used as secondary antibodies. The presence of antibodies on the PVDF membranes was detected via treatment with Pierce ECL western blotting substrate (Thermo Scientific) and captured using the Syngene XR5 gel documentation system. Protein levels were assessed using Image J (NIH). The SMC3 Co-IP experiment was performed using the Dynabead® Co-IP kit (Life Technologies). Each milligram of beads was covalently linked to 4 µg of SMC3 antibody (Abcam, ab9263) or corresponding IgG control antibody (Life Technologies, A10533).
Trans blot turbo western transfer system
Manufactured by Bio-Rad
The Trans-Blot Turbo Western transfer system is a laboratory equipment used for the rapid and efficient transfer of proteins from polyacrylamide gels to membranes, a critical step in Western blotting analysis. The system utilizes a high-powered electrical current to facilitate the transfer process, allowing for faster and more consistent results compared to traditional wet transfer methods.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (3 mentions)
Ripa buffer, by Santa Cruz Biotechnology (3 mentions)
Protease inhibitor cocktail, by Roche (3 mentions)
Gradient sds polyacrylamide gel, by Bio-Rad (3 mentions)
Goat anti mouse 62 6520, by Thermo Fisher Scientific (2 mentions)
Goat anti rabbit a10533 horseradish peroxidase conjugated antibodies, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Ab9263, by Abcam (1 mentions)
3 protocols using trans blot turbo western transfer system
1
Western Blot Analysis of Protein Levels
2
Protein Extraction and Analysis via SDS-PAGE and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
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For protein level analyses, proteins were extracted from germ cells using RIPA buffer (Santa Cruz) containing 1× protease inhibitor cocktail (Roche). Protein concentration was calculated using a Bicinchoninic acid (BCA) protein assay kit (Pierce). Lanes of 6%, 10%, 15%, and 4–15% gradient SDS polyacrylamide gels (Bio-Rad) were loaded with 20 µl of 1 mg/ml protein extract. Following protein separation via standard SDS–PAGE, proteins were transferred to polyvinylidene difluoride (PVDF) membranes using the Trans-Blot Turbo Western transfer system (Bio-Rad). Primary antibodies and dilution used are presented in Supplemental Table S2. At a 1:5000 dilution, goat anti-mouse (62-6520) and goat anti-rabbit (A10533) horseradish peroxidase–conjugated antibodies (Invitrogen) were used as secondary antibodies. The presence of antibodies on the PVDF membranes was detected via treatment with Pierce ECL Western blotting substrate (Thermo Scientific) and captured using the Syngene XR5 gel documentation system. Protein levels were assessed using Image J (NIH).
3
Western Blot Analysis of Germ Cell Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For protein level analyses, proteins were extracted from germ cells using RIPA buffer (Santa Cruz) containing 1× protease inhibitor cocktail (Roche). Protein concentration was calculated using a BCA protein assay kit (Pierce). Lanes of 4–15% gradient SDS polyacrylamide gels (Bio-Rad) were loaded with 20 µl of 1 mg/ml protein extract. For STA-PUT, 20 µl of 0.1 mg/ml protein extracts from purified leptotene/zygotene and pachytene/diplotene stage spermatocytes and round spermatids were loaded per lane on SDS polyacrylamide gels. Following protein separation via standard SDS–PAGE, proteins were transferred to polyvinylidene difluoride (PVDF) membranes using the Trans-Blot Turbo Western transfer system (Bio-Rad). Primary antibodies and dilution used are presented in Supplemental Table S2. At a 1:5000 dilution, goat anti-mouse (62-6520) and goat anti-rabbit (A10533) horseradish peroxidase–conjugated antibodies (Invitrogen) were used as secondary antibodies. The presence of antibodies on the PVDF membranes was detected via treatment with Pierce ECL Western blotting substrate (Thermo Scientific) and captured using the Syngene XR5 gel documentation system. Protein levels were assessed using Image J (NIH).
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