Cells were washed twice with cold PBS. Then the cells were lysed with RIPA lysis buffer (50 × 10−3m Tris‐HCl (pH 7.4), 150 × 10−3m NaCl, 1% Triton X‐100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, leupeptin) for 30 min. The cell lysate was centrifuged at 12 000 rpm for 15 min. The protein concentration was measured using BCA protein assay kit. Equal amounts of total protein were separated on 10% or 15% SDS‐PAGE and transferred onto NC membranes, blocked with 5% nonfat milk at room temperature for 1 h, and incubated with primary antibodies at 4 ˚C overnight. After washed three times, the membranes were incubated with secondary Ig conjugated HRP for 1 h at room temperature. Protein bands were visualized with the ECL enhanced chemiluminescence regent Kit (NCM Biotech). The following antibodies were used: RBMS1 (Abcam ab150353), S100P (Proteintech 11803‐1‐AP), YTHDF1 (Proteintech 17479‐1‐AP), YTHDF2 (Proteintech 24744‐1‐AP), YTHDF3 (Proteintech 25537‐1‐AP), METTL3 (Proteintech 15073‐1‐AP), METTL14 (Proteintech 26158‐1‐AP), FTO (Proteintech 27226‐1‐AP), m6A (Proteintech 68055‐1‐lg), FLAG (Sigma 1804), V5 (Proteintech 14440‐1‐AP), VINCULIN (Proteintech 66305‐1‐Ig), GAPDH (Proteintech 60004‐1‐Ig).
Rbms1
Manufactured by Abcam
RBMS1 is a protein that functions as an RNA-binding protein. It is involved in the regulation of mRNA metabolism and transport.
Automatically generated - may contain errors
Lab products found in correlation
Flag tag (flag), by Merck Group (1 mentions)
Ythdf2, by Proteintech (1 mentions)
Mettl14, by Proteintech (1 mentions)
Vinculin, by Proteintech (1 mentions)
Ythdf1, by Proteintech (1 mentions)
Ythdf3, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Mettl3, by Proteintech (1 mentions)
Bradford assay, by Thermo Fisher Scientific (1 mentions)
Gap43, by Merck Group (1 mentions)
Wave1, by Abcam (1 mentions)
Gm130, by BD (1 mentions)
Ab150353, by Abcam (1 mentions)
Cpeb4, by Abcam (1 mentions)
2 protocols using rbms1
1
Western Blotting of m6A Regulators
2
Standardized Western Blot Technique
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Western blots were performed using standard tris-glycine SDS-PAGE protocols. Samples had normalized protein concentration assessed using a Bradford assay (#23246, Thermo). Resolved proteins were electroblotted onto PVDF membranes using semi-dry transfer, blocked for 30 min in TBS supplemented with 0.1% Tween-20 and 5% skim milk, and incubated with primary antibodies (GAP43 #MAB347 Millipore; Actb #A5441 Sigma; Gm130 #610823 BD Biosciences; MAP2 #M1406 Sigma; WAVE1 #ab272916 abcam; WAVE2 #3659T Cell Signaling Technologies; WAVE3 #2806S Cell Signaling Technologies; Cpeb4 #ab224162 abcam; Rbms1 #ab150353 abcam; Tau #AF3494 RR&D) in TBS supplemented with 0.1% Tween-20 and 3% BSA over night at 4°C. The next day, the membrane was washed three times in TBS supplemented with 0.1% Tween-20, incubated for 2 hours at room temperature with secondary antibodies that were isotype-specific, HRP-conjugated, and cross-absorbed (Life Technologies), then washed again three times with TBS supplemented with 0.1% Tween-20. Immunoreactive bands were visualized through detection of chemiluminescence using a CCD camera imager (FluoroChemM, Protein Simple). Quantification of signal intensity in the various bands was performed using the standard Fiji software package.
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