Reversible Negative Staining of Protein on Electrophoresis Gels
35
2.4
Transfer to Membranes
2.4.1 Gel Blotting
Procedure
1. Apply the procedure for protein mobilization (2.3) to the entire gel.
2. Wash the gel with abundant MQW (2 x 1 min).
3. Follow the established procedures for semi-dry blotting to PVDF or nitrocellulose membranes (0.8 mA/cm2, for 40-60 mins).
4. After blotting, wash the membrane with abundant MQW and detect as usual.
2.4.2
Single Band Transfer (Fernandez-Patron et al 7995)
Procedure
1. Follow the procedure described under 2.4.1 for gel transfer with the following
modifications: Excise the band of interest, mobilize the protein (see 2.3) and
wash with water (2 x 1 mL x 1 min).
2. Cut the blotting paper and the transfer membrane to fix the size of the excised
gel band.
3. Mount and place the individual mini-sandwiches at the center of the semi-dry
graphite electrode. Teflon spacers can be placed on the lower electrode at both
sides of the mini-sandwiches to afford an adequate stability of the upper electrode (the height of the spacers should be the same or slightly lower than that
of the mini-sandwiches).
4. Transfer at 0.8 mA/cm 2 for 40-60 mins.
Comments
i) Ready-to-use membrane disks furnished with the sequencers can be used for
mounting the transfer.
ii) When working with small amounts of proteins, several (2-3) tiny bands
excised from different lanes can be placed on a single membrane disk, to
increase the amount of protein on the blot.
Advantages
Single band transfer allows for optimized transfer conditions (blotting buffers
and transfer time) for each protein of interest in the gel, with respect to its electrophoretic mobility. Sensitivity of protein detection on the gel by Reversestaining is higher than that attained with amido black on the membrane after
transfer; this allows for sequence information to be obtained from protein
amounts that were previously detectable on the gel but insufficient for detection
on the membrane after blotting. In summary, once the conditions for transfer
have been established, staining of the membrane can be obviated and the membrane can be directly analyzed, thus, avoiding artifacts from the staining reagent.
35
2.4
Transfer to Membranes
2.4.1 Gel Blotting
Procedure
1. Apply the procedure for protein mobilization (2.3) to the entire gel.
2. Wash the gel with abundant MQW (2 x 1 min).
3. Follow the established procedures for semi-dry blotting to PVDF or nitrocellulose membranes (0.8 mA/cm2, for 40-60 mins).
4. After blotting, wash the membrane with abundant MQW and detect as usual.
2.4.2
Single Band Transfer (Fernandez-Patron et al 7995)
Procedure
1. Follow the procedure described under 2.4.1 for gel transfer with the following
modifications: Excise the band of interest, mobilize the protein (see 2.3) and
wash with water (2 x 1 mL x 1 min).
2. Cut the blotting paper and the transfer membrane to fix the size of the excised
gel band.
3. Mount and place the individual mini-sandwiches at the center of the semi-dry
graphite electrode. Teflon spacers can be placed on the lower electrode at both
sides of the mini-sandwiches to afford an adequate stability of the upper electrode (the height of the spacers should be the same or slightly lower than that
of the mini-sandwiches).
4. Transfer at 0.8 mA/cm 2 for 40-60 mins.
Comments
i) Ready-to-use membrane disks furnished with the sequencers can be used for
mounting the transfer.
ii) When working with small amounts of proteins, several (2-3) tiny bands
excised from different lanes can be placed on a single membrane disk, to
increase the amount of protein on the blot.
Advantages
Single band transfer allows for optimized transfer conditions (blotting buffers
and transfer time) for each protein of interest in the gel, with respect to its electrophoretic mobility. Sensitivity of protein detection on the gel by Reversestaining is higher than that attained with amido black on the membrane after
transfer; this allows for sequence information to be obtained from protein
amounts that were previously detectable on the gel but insufficient for detection
on the membrane after blotting. In summary, once the conditions for transfer
have been established, staining of the membrane can be obviated and the membrane can be directly analyzed, thus, avoiding artifacts from the staining reagent.