Reduced protein extracts (10–20 μg protein) or immunoprecipitates were resolved on either 8% (v/v) SDS-PAGE gels, or 4-12% NuPAGE bis-tris precast gradient gels (Invitrogen) by electrophoresis. Separated proteins were subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore), before membranes were blocked in 5% (w/v) non-fat milk powder (Marvel) in TBS-T (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2% (v/v) Tween-20) and incubated overnight at 4°C in 5% milk TBS-T or 5% bovine serum albumin (BSA) TBS-T with the appropriate primary antibody. Membranes were then washed 3 X 10 min with TBS-T before incubating with HRP-conjugated secondary antibodies in 5% milk TBS-T for 1 h at room temperature. Membranes were then washed 3 X 10 min with TBS-T before detection with enhanced chemiluminescence reagent (Millipore) and exposure to medical-grade X-ray films (Konica Minolta), as described previously (2 , 47 (link), 48 (link)).
Nupage bis tris precast gradient gels
Manufactured by Thermo Fisher Scientific
NuPAGE Bis-Tris precast gradient gels are polyacrylamide gel electrophoresis (PAGE) products designed for the separation and analysis of proteins. These gels feature a Bis-Tris buffer system and a gradient of polyacrylamide concentrations to enable efficient protein separation and resolution.
Automatically generated - may contain errors
2 protocols using nupage bis tris precast gradient gels
1
Western Blot Protein Analysis Protocol
2
Western Blot Analysis of Protein Extracts
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Reduced protein extracts (typically 10–20 μg protein) or IPs were resolved on either 8 or 13% (v/v) SDS–PAGE gels, or 4–12% NuPAGE bis‐tris precast gradient gels (Invitrogen) by electrophoresis. Separated proteins were subsequently transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore), before membranes were blocked in 5% (w/v) non‐fat milk powder (Marvel) in TBS‐T (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.2% (v/v) Tween‐20) and incubated overnight at 4°C in either 5% milk TBS‐T or 5% bovine serum albumin (BSA) TBS‐T with the appropriate primary antibody. Membranes were then washed for 3 × 10 min with TBS‐T before incubating with HRP‐conjugated secondary antibodies in 5% milk TBS‐T for 1 h at RT. Membranes were then washed for 3 × 10 min with TBS‐T before detection with enhanced chemiluminescence reagent (Millipore) and exposure to medical‐grade X‐ray films (Konica Minolta), as described previously 20 , 59 , 61 . Alternatively, membranes were imaged using the ChemiDoc™ system (Bio‐Rad).
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