UWB1.289 and A2780 cells were counted, and 106 cells were mixed with 1 ml RIPA (GenePharma) to extract total proteins. Proteins were boiled in water for 5 min, and electrophoresis was performed using 10% SDS-PAGE gel. PVDF membranes were used in gel transfer, and PBS containing 5% non-fat milk was used to block at room temperature for 2 h. GAPDH (1:900, ab37168, Abcam) and snail (1:900, ab53519, Abcam) primary antibodies were used in the first blot, which was performed at 4 °C overnight. HRP goat anti-rabbit (IgG) (1:1000; ab6721; Abcam) secondary antibody was used in the second blot, which was performed at room temperature for 2 h. RapidStep™ ECL detection reagent (EMD Millipore) was dropped onto the membranes to develop signals. Gray values were analyzed using Image J v1.46 software.
Radioimmunoprecipitation assay (ripa)
Manufactured by GenePharma
RIPA is a widely used cell lysis buffer for protein extraction and purification. It effectively disrupts cell membranes and solubilizes proteins, making it suitable for a variety of downstream applications such as Western blotting, immunoprecipitation, and enzyme-linked immunosorbent assays (ELISA).
Automatically generated - may contain errors
Lab products found in correlation
Rapidstep ecl detection reagent, by Merck Group (1 mentions)
Ab37168, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
Ab53519, by Abcam (1 mentions)
Stat6, by Abcam (1 mentions)
P stat6, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
P akt, by Abcam (1 mentions)
E cadherin, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
β actin, by Abcam (1 mentions)
Tsg101, by Abcam (1 mentions)
Calnexin, by Abcam (1 mentions)
2 protocols using radioimmunoprecipitation assay (ripa)
1
Quantifying Protein Expression in Cancer Cells
2
Western Blot Analysis of Exosomal Proteins
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RIPA (Genepharma) was used for extracting total protein. BCA was used for total protein quantification. SDS-PAGE (10%) was used to separate the proteins. The proteins were then transferred onto PVDF membranes (MilliporeSigma). Primary antibodies targeted against CD63 (cat. no. ab134045, 1:1,000; Abcam, Cambridge, MA, USA), TSG101 (cat. no. ab125011, 1:1,000; Abcam), p-STAT6 (cat. no. ab263947, 1:1,000; Abcam), STAT6 (cat. no. ab32108, 1:1,000; Abcam), N-cadherin (cat. no. ab76011, 1:1,000; Abcam), E-cadherin (cat. no. ab40772, 1:1,000; Abcam), calnexin (cat. no. ab133615, 1:1,000; Abcam), PTEN (cat. no. ab267787, 1:1,000; Abcam), Akt (cat. no. ab8805, 1:1,000; Abcam), p-Akt (cat. no. ab38449, 1:1,000; Abcam) and β-actin (cat. no. ab8226, 1:1,000; Abcam) were used to incubate the membranes at 4°C overnight after blocking for 1 h with skimmed milk (5%). HRP-conjugated secondary antibodies (cat. no. ab288151, 1:5,000; Abcam) were used to incubate the membranes. The protein bands were visualized using ECL (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). IPP 6.0 (Image-Pro Plus 6.0) was applied for densitometric analysis.
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