Hi folks,
Which protein transfer method is better for low MW proteins (8KDa in this case)? I have heard wet transfer is better because it ensures complete transfer but I've only been getting faint protein bands with wet. I do the transfer at 100V for 1 hour using the bio-rad wet transfer tank.
Many thanks in advance.
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#2
Inmost sun
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Posted 18 June 2010 - 09:57 AM
In_search_of_truth, on Jun 18 2010, 04:26 PM, said:
Hi folks,
Which protein transfer method is better for low MW proteins (8KDa in this case)? I have heard wet transfer is better because it ensures complete transfer but I've only been getting faint protein bands with wet. I do the transfer at 100V for 1 hour using the bio-rad wet transfer tank.
Many thanks in advance.
Which protein transfer method is better for low MW proteins (8KDa in this case)? I have heard wet transfer is better because it ensures complete transfer but I've only been getting faint protein bands with wet. I do the transfer at 100V for 1 hour using the bio-rad wet transfer tank.
Many thanks in advance.
semi-dry blotting will do
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Posted 18 June 2010 - 09:58 AM
I've heard the opposite - that wet WB is better for high MW proteins and semi-dry is better for low MW proteins, can't find the source though...
I've used semi-dry with Towbin buffer (Tris-Gly) or Dunn buffer (carbonate), 0.22um Immobilon Psq membrane, 0.8mA/cm2 for 1 hour and got quite nice results for 6-8 kDa proteins.
I've used semi-dry with Towbin buffer (Tris-Gly) or Dunn buffer (carbonate), 0.22um Immobilon Psq membrane, 0.8mA/cm2 for 1 hour and got quite nice results for 6-8 kDa proteins.
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Posted 18 June 2010 - 01:10 PM
K.B., on Jun 18 2010, 10:58 AM, said:
I've heard the opposite - that wet WB is better for high MW proteins and semi-dry is better for low MW proteins, can't find the source though...
I've used semi-dry with Towbin buffer (Tris-Gly) or Dunn buffer (carbonate), 0.22um Immobilon Psq membrane, 0.8mA/cm2 for 1 hour and got quite nice results for 6-8 kDa proteins.
I've used semi-dry with Towbin buffer (Tris-Gly) or Dunn buffer (carbonate), 0.22um Immobilon Psq membrane, 0.8mA/cm2 for 1 hour and got quite nice results for 6-8 kDa proteins.
Hi K.B, thanks very much for your help. I'm new to the world of protein gels!
Ok I'll try the semi-dry. But please can you tell me what constant current you run yours at (say for a single mini gel if that changes anything)? I use the 0.2um nitrocellulose membrane and we have got the 'standard' bio-rad semi dry transfer set up. Also if you don't mind can you check if the recipe below is ok for the semi-dry transfer buffer,
Tris 2.91g
Glycine 1.465g
SDS 1.875ml
100ml of Meoh
---->>> made up to 500ml with dH20
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Posted 14 July 2010 - 01:08 AM
In_search_of_truth, I'm sorry for the delay but I was quite busy with other stuff...
I use current 0.8mA per square centimetre of membrane surface - you need to calculate correct value for size of your membrane eg. 10cm x 10cm = 100 cm2 x 0.8 mA = 80 mA = 0,08 A
As for transfer buffers - I use:
Towbin buffer: 0.192 M Gly + 25 mM Tris + 20% MeOH
Dunn carbonate buffer: 10 mM NaHCO3 + 3 mM Na2CO3 + 20% MeOH
I'm not an expert but I think you should skip SDS in your transfer buffer when working with low MW proteins.
I use current 0.8mA per square centimetre of membrane surface - you need to calculate correct value for size of your membrane eg. 10cm x 10cm = 100 cm2 x 0.8 mA = 80 mA = 0,08 A
As for transfer buffers - I use:
Towbin buffer: 0.192 M Gly + 25 mM Tris + 20% MeOH
Dunn carbonate buffer: 10 mM NaHCO3 + 3 mM Na2CO3 + 20% MeOH
I'm not an expert but I think you should skip SDS in your transfer buffer when working with low MW proteins.
