1- In order to eliminate any risks of contaminations, use clean trays. In particular do not share trays and glassware that are used for western blotting and immunodetection as they are contaminated with large amounts of proteins (BSA, caseins, etc.).

2- In order to minimize blockage of the N-terminal amine of your protein: 

  • Use fresh solutions. 
  • Use high purity gel reagents. 
  • Let the gel sit prior to running your protein to allow for complete polymerization. 
  • Use a mini-gel system with loading as much protein as possible.

3- Optimisation of transfer conditions Electroblotting is the most critical step in obtaining sufficient amounts of protein on the membrane. Careful optimization of the amperage and transfer time is important for the highest recovery of your protein. Generally speaking, lower current yields more efficient transfers, but requires longer times. The table below indicates approximate transfer time from mini-gel.

Protein size (kDa)
Transfer time (min) *
15-20 kDa
10-15 min
100 kDa
45-60 min

(*) Current set at 0.5 mA.