Western botting was performed as previously described [17 (link)]. Briefly, cells pellets were lysed on ice for in EBC buffer (50 nM Tris–HCl pH 8.0, 130 mM NaCl, 1% Triton X-100, 0.1% SDS) supplemented with protease inhibitor cocktail (Sigma, Milan-Italy). Cell lysates were separated by 10% SDS-PAGE gel followed by transfer to Hybond ECL nitrocellulose membrane (GE Healthcare, Milan, Italy). Secondary antibodies conjugated with HRP were used at a dilution of 1:5000 and the reaction was revealed using the ECL Western Blotting Kit (Promega, Milan-Italy) according to the manufacturer’s instructions. Antidody dilutions are reported in Table 1 . Secondary antibodies conjugated with HRP were used at a dilution of 1:5000 and the reaction was revealed using the ECL Western Blotting Kit (W1001, Promega, Milan-Italy) according to the manufacturer’s instructions.
Ecl western blotting kit
Manufactured by Promega
Sourced in Italy
The ECL Western Blotting Kit is a laboratory equipment product designed for the detection and analysis of proteins using the Western blotting technique. It provides the necessary reagents and components for the chemiluminescent detection of target proteins on a membrane after electrophoretic separation and transfer.
Automatically generated - may contain errors
Lab products found in correlation
Hybond ecl nitrocellulose membrane, by GE Healthcare (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Lysis buffer, by Biosharp (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Anti drosha antibody, by Abcam (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Anti β actin antibody, by Abcam (1 mentions)
Non fat milk, by Merck Group (1 mentions)
Anti dnmt3a, by Abcam (1 mentions)
Anti actin, by BD (1 mentions)
3 protocols using ecl western blotting kit
1
Western Blotting Protocol for Protein Detection
2
DROSHA Protein Expression Analysis
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Lysis buffer (Biosharp, Hefei, China) including 5 g/L sodium deoxycholate, 0.2 g/L NaN3,10 mL/l NP-40, 150 mmol/L NaCl 100 μg/mL, 0.1 g/LSDS, phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, and 50 mmol/L Tris-HCl (pH 8.5) was used to lyse the cells or tissues according to the manufacturer’s instructions. We used 12% SDS-PAGE to purify protein samples and then transferred them to nitrocellulose membranes (GE Healthcare, Milan, Italy). To avoid unspecific binding, Tris-buffered saline/Tween-20 (0.1%, Bioeasy, Shanghai, China) including 5% non-fat milk (Merck, Darmstadt, Germany) was used to incubate with the membrane at room temperature for 2 h. Subsequently, monoclonal antibodies against DROSHA (anti-DROSHA antibody, 1:1000, RT, 2h, Abcam, Boston, MA) were incubated with the blot, and at the same time a monoclonal antibody against β-actin (anti-β-actin antibody, 1:10000, RT, 1 h, Abcam, Boston, MA) were used as an internal control. After washing with PBS (Invitrogen, CA), secondary antibody conjugated to HRP (1:10000, RT, 1 h, Abcam, Boston, MA) and the ECL Western Blotting Kit (Promega, Milan, Italy) were used for signal detection according to the manufacturers’ protocols.
3
Western Blot Analysis of DNA Methyltransferases
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Cells pellets were lysed on ice for in EBC buffer (50 nM Tris–HCl pH 8.0, 130 mM NaCl, 1% Triton X-100, 0.1% SDS) supplemented with protease inhibitor cocktail (Sigma, Milan-Italy). Cell lysates were separated by 10% SDS-PAGE gel followed by transfer to Hybond ECL nitrocellulose membrane (GE Healthcare, Milan, Italy). Western blotting was made using anti-DNMT1 (1:400, BD, NJ USA), anti-DNMT3a (1:250, Abcam, UK) and anti-actin (1:1000, BD, NJ USA). Secondary antibodies conjugated with HRP were used at a dilution of 1:5000 and the reaction was revealed using the ECL Western Blotting Kit (Promega, Milan-Italy) according to the manufacturer’s instructions.
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