Guide to successful electroblotting to PVDF - use Immobilon PSQ or ProBlott

1. Choose a gel system which gives good separation of the desired protein bands; a band running in the lower half of the gel will blot better than if it is in the top half so adjust acrylamide concentration accordingly.

2. Avoid urea in the gel if possible - it can cause N-blocks at alkaline pH.

3. Include 2mM-mercaptoacetic acid (thioglycollic acid) in the upper electrode buffer. This mobile thiol will run ahead of the protein and scavenge N-blocking free-radicals.

4. Use thin gels and ensure acrylamide is of high quality and deionised before use.

5. Where possible, cast gels a day before use to allow the polymerisation to go to completion - free acrylamide can N-block proteins - or use precast gels.

6. Using 100-200pmol of each protein in one or two tracks of the gel, run the gel and then soak it, with shaking, in transfer buffer for 15min. If poor transfer is experienced, increasing the equilibration time to 30min can be beneficial. Transfer buffers commonly used are 25mM Trizma, 190mM glycine, 10% MeOH; 10mM CAPS, 5mM DTT, 10% MeOH, pH 11; 50mM N-ethylmorpholine pH 8.3, 10% MeOH, 5mM DTT. The Trizma buffer is suitable for most applications where the proteins of interest are below 50-60kDa. The inclusion of SDS is not usually necessary, but for difficult proteins (e.g. pI>7) the presence of no more than 0.02% SDS can be helpful. Higher amounts of SDS can reduce the binding capacity of PVDF for protein. The CAPS buffer is generally preferable for high Mr proteins (e.g. >50 000). For difficult membrane proteins, the inclusion of 0.05% Triton X-100 or Tween in the equilibration buffer only can help keep the protein soluble; omit the detergent from the transfer buffer.

7. Where problems of N-blocking are encountered, the use of a lower pH gel system can be advantageous (see Moos et al. (1988) J.Biol. Chem. 263, 6005-6008). However, if protein identification rather than N-terminal analysis is important, alternative approaches (proteomics) can solve the problem.

8. N.B. All washing/equilibration steps must be done with gentle agitation or shaking. Wearing gloves at all times when handling PVDF, prepare the PVDF membrane by immersion in AR MeOH for 10 sec followed by equilibration in transfer buffer for 5min. Prepare a gel/PVDF/blotting paper sandwich as for Western blotting, ensuring exclusion of all air bubbles. When blotting onto PVDF for the first time with a given protein whose behaviour on electroblotting is unknown, the inclusion of 2 membranes on the anodic side of the gel and 1 membrane on the cathodic side will monitor excessive transfer or the presence of extremely electropositive proteins respectively.

9. Conduct the electroblotting using conditions similar to those used for Western blotting to nitrocellulose (but note that the MeOH concentration is typically lower). In wet-blots, 300mA for 1-3h is generally sufficient although the transfer time and current must be optimised for each protein studied. Excessive transfer time (e.g. overnight) can be detrimental and insufficient transfer leads to poor yield. In this respect, it is advisable to optimise the transfer of related proteins before submitting the protein under study should this be in short supply. In semi-dry blots, 0.8-1mA/cm2 for 30min-1.5h is usually satisfactory.

10. When transfer is thought to be complete, remove the PVDF membrane and wash immediately in water for 10min with shaking. If the membrane is allowed to dry at any stage, even partially, it must be re-wet with MeOH before immersion in aqueous medium.

Immobilon P - Stain the membrane with 0.1% Coomassie Blue R in 50% MeOH for 5min and destain with several changes, 2-5min each, of 50% MeOH, 10% acetic acid. We recommend using Immobilon PSQ or ProBlott - these supports give better results.

Immobilon PSQ or Problott - Stain the membrane with 0.1% Coomassie Blue R in 50% MeOH, 1% acetic acid for 5min and destain with several changes, 2-5min each, of 50% MeOH. The membrane will remain a faint blue colour; do not try to make it white!
Finally wash the membrane in water (2x5min, shaking) and air-dry thoroughly; damp membranes will not be accepted for sequence analysis. Place the membrane in a plastic bag and store dry at 4^oC.
If your protein does not react with Coomassie, Amido Black and Ponceau S stains are compatible with sequence analysis, although they are less sensitive stains.