I want to transfer a 245kDA protein . I am using 8% gel and PVDF membrane. What are the semi dry conditions that i should use to achieve a complete transfer
What type of gel (hand or precast)? The firm of blotting instrument?
Neha Gupta
SDS-PAGEs are not a good choice for blotting HMW proteins. So, in this case you have to apply a special blotting strategy. Some tips help to blot the HMW proteins efficiently:
1. Omit or reduce the percentage of Methanol.
2. Add SDS up to 0.05% to the blotting buffer.
3. Don not equilibrate the gel in a blotting buffer that contains methanol neither ethanol after gel electrophoresis.
4. If you do not have to detect LMW proteins, use 0.45 PVDF.
5. Use tris-acitate (3-8%) gels.
Pls refer to the following article which suggested a new protocol for efficient blotting of HMW protein:
- DOI: 10.1007/978-1-59745-542-8_20
Hi Neha
Beside what Mohamed Khashan mentioned I would like to add that you might want to try methanol at 5% or even omit it completely.
For Turboblot system (from BIORAD), I remember detecting mTOR and p-mTOR. The conditions that i used were as follows:
1.3Ampere, 25V for 25 minutes.
Please use chilled transfer buffer if you haven't been using already.
Since we used NC membrane instead of PVDF, so we never activated it in methanol prior to transfer and hence this may be one of the factors that you also want to pay attention to.
Hope it helps
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