The soluble and insoluble cell lysate fractions were evaluated by 12% polyacrylamide gel electrophoresis under denaturing conditions (SDS-PAGE) (Coomassie blue staining) [18] . A replica of the gel was transferred to a nitrocellulose membrane (0.45 µm thick; ThermoScientific) in transfer buffer (25 mM Tris base, 192 mM glycine, and 10% methanol v/v) at 200 mA for 2 h. The membrane was blocked overnight with a 5% w/v skim milk solution in TBST. Immunodetection of the recombinant protein was performed using a histidine tag with the primary antibody anti-His-mAb (1:3000, Amersham) and secondary antibody anti-mAb-HRP (1:2000); 4-chloro-naphthol (Sigma) and hydrogen peroxide were used as developing agents [19] .
4 chloro naphthol
Manufactured by Merck Group
Sourced in United States
4-chloro-naphthol is a chemical compound used in various laboratory applications. It is a crystalline solid that is soluble in organic solvents. The compound is often utilized as a reagent or intermediate in chemical synthesis and analysis processes.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Gel pro4, by Media Cybernetics (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Gapdh antibody, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
2 protocols using 4 chloro naphthol
1
SDS-PAGE and Immunoblot Analysis of Recombinant Protein
2
Western Blot Analysis of Apoptotic Markers
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Total protein was extracted in lysis buffer containing PMSF (Pierce; Rockford, IL). The proteins were separated in a 10% SDS-PAGE and immunoblotted onto polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, USA). Non-specific binding to the membranes was blocked by incubation with 5% nonfat dried milk for 1–2 h, and the membranes were probed separately with rabbit anti-human Bax, Bcl-2, COX-2, cleaved caspase-3, and GAPDH antibodies (1:100, Invitrogen) overnight at 4 °C, followed incubation with the appropriate horseradish peroxidase-conjugated secondary antibody (1:10000, Invitrogen) for 1 h; GAPDH was used as the internal control. The protein bands were visualized by the application of 4-chloronaphthol (Sigma-Aldrich) and analyzed by Gel-Pro 4.0 software (Media Cybernetics, Inc., USA).
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