Western blot analysis was performed, as described elsewhere [8] . Briefly, at least 16 pooled organoids per each condition were washed three times with PBS (pH 7.4) and lysed in buffer containing 50 mM Tris-HCl (pH 6.8), 10% glycerol and 1% sodium dodecyl sulfate (SDS). The lysates were homogenized by sonication and protein concentrations were determined using DC Protein Assay (Bio-Rad). The lysates were then supplemented with 0.01% bromophenol blue and 1% β-mercaptoethanol and denatured at 100 °C for 5 minutes. The prepared samples were separated by SDSpolyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore). The PVDF membrane was blocked in 5% skimmed milk in Tris-buffered saline containing Tween for 1 hour and incubated with primary antibodies overnight at 4 °C. The following antibodies were used: PAX6 (sc-53108, Santa Cruz, 1:1000), SOX2 (sc-365823, Santa Cruz, 1:1000), SOX1 (bs-12276R, Bioss, 1:1000), β-ACTIN (#4970S, Cell Signaling Technology, 1:1000).
Bs 12276r
Manufactured by Bioss Antibodies
The Bs-12276R is a piece of laboratory equipment. It is designed for general laboratory use. The core function of the Bs-12276R is to [description not available].
Automatically generated - may contain errors
2 protocols using bs 12276r
1
Western Blot Analysis of Organoid Markers
2
Western Blot Analysis of Organoid Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Western blot analysis was performed, as described elsewhere [8] . Briefly, at least 16 pooled organoids per each condition were washed three times with PBS (pH 7.4) and lysed in buffer containing 50 mM Tris-HCl (pH 6.8), 10% glycerol and 1% sodium dodecyl sulfate (SDS). The lysates were homogenized by sonication and protein concentrations were determined using DC Protein Assay (Bio-Rad). The lysates were then supplemented with 0.01% bromophenol blue and 1% β-mercaptoethanol and denatured at 100 °C for 5 minutes. The prepared samples were separated by SDSpolyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore). The PVDF membrane was blocked in 5% skimmed milk in Tris-buffered saline containing Tween for 1 hour and incubated with primary antibodies overnight at 4 °C. The following antibodies were used: PAX6 (sc-53108, Santa Cruz, 1:1000), SOX2 (sc-365823, Santa Cruz, 1:1000), SOX1 (bs-12276R, Bioss, 1:1000), β-ACTIN (#4970S, Cell Signaling Technology, 1:1000).
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