3 replies to this topic

[science noob]

Hi,

I have been trying to transfer a 400kDa protein on 6% gel where I ran the dye front + an additional 30-60 min to run off smaller sized proteins with reference to a protein ladder.
What I have observed is the protein ladder (PAGE RULER) starts to become fainter with time. Does this mean that the proteins are "leaking" out?

I've added 0.1% SDS and 20% Methanol into my Tris-Glycine transfer buffer.  Transfer was ran overnight @ 20V.

My observation is that the bands on the protein ladder doesn't seem to be as clear as when I transferred smaller proteins.

Has anyone successfully transferred such a large protein before? And what can I modify to get a better blot?

[mdfenko]

first, you shouldn't use more than 0.05% sds in the transfer buffer.

is your protein ladder prestained? are you seeing the stain decrease? mostly in the lower sizes or equally with the larger sizes?

to what pore size membrane are you transferring? for prolonged transfer times you may want to consider using a smaller pore membrane (eg 0.2um) to reduce "blow-through" of the proteins.

[science noob]

mdfenko, on 22 November 2011 - 06:58 AM, said:

first, you shouldn't use more than 0.05% sds in the transfer buffer.

is your protein ladder prestained? are you seeing the stain decrease? mostly in the lower sizes or equally with the larger sizes?

to what pore size membrane are you transferring? for prolonged transfer times you may want to consider using a smaller pore membrane (eg 0.2um) to reduce "blow-through" of the proteins.

1. What will a >0.05% SDS content do to the transfer?

2. Protein ladder was prestained.  Stain certainly decreased with time of running the SDS-PAGE, all sizes became fainter, lower sizes fade more than larger ones.

3. I was using an Immobilon-P PVDF membrane (0.45um).  Wouldn't a smaller pore sized membrane reduce the efficiency of large protein transfer?

[mdfenko]

science noob, on 22 November 2011 - 07:21 PM, said:

1. What will a >0.05% SDS content do to the transfer?

too much sds in the transfer buffer will interfere with binding to the membrane. it will reduce contact with the membrane by getting in the way and will cause the protein to migrate to quickly for capture. the methanol in the transfer buffer is also present to strip the sds from the protein.

Quote

2. Protein ladder was prestained.  Stain certainly decreased with time of running the SDS-PAGE, all sizes became fainter, lower sizes fade more than larger ones.

during the page run? or the transfer?

if during the page run then it is caused by diffusion of the band. this can be reduced with proper stacking and/or using a gradient gel.

if during transfer then it may be due to blow through or electrolytic stripping of the dye (not likely because the dye should be covalently bound).

Quote

3. I was using an Immobilon-P PVDF membrane (0.45um).  Wouldn't a smaller pore sized membrane reduce the efficiency of large protein transfer?

no, it will actually improve binding efficiency (within the capacity of the membrane) because the protein won't blow through as easily.

Edited by mdfenko, 23 November 2011 - 09:14 AM.