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)).
Medical grade x ray films
Manufactured by Konica Minolta
Medical-grade X-ray films are photographic films used in the medical imaging industry to capture and record X-ray images. These films are specifically designed to meet the high-quality and performance standards required for diagnostic medical applications.
Automatically generated - may contain errors
2 protocols using medical grade x ray films
1
Western Blot Protein Analysis Protocol
2
Western Blot Analysis of Protein Extracts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!