Western Blot - Semidry Transfer (Feb/05/2005 )
Dear All
Please solve my problem
I am facing the problem in transfering of protein (25-50kd) from SDS page to PVDF membrabne with biorad semidry instrument. First time i got result latter on i am not getteing result.
Transfer Buffer
25mM Tris
192mM Glycin
0.037% SDS
methanol 20 % for i litre. And Transfer at 15 volt for 2 hr.
Where i am doing mistake please traceout.
If Any one have solution please help me
hi
first of all try not use sds for your transfert buffer !
second one, take care not do get more than 15volts;
finally check if your pvdf membrane supports methanol. Some memebranes do not support methanol.
keep us informed 
my buffers (found in maniatis):
running :
glycine 1,92M
SDS 1%
tris base 250mM
for one liter, it is 144.13g 10g and 30,28g respectively
transfert :
glycine 1,92M
tris base 250mM
for one liter, it is 144.13g and 30,28g respectively
If you want, you can add methanol :
10X transfert solution 100 ml
methanol 200ml
ddh2O qsp 1 liter.
first of all try not use sds for your transfert buffer !
second one, take care not do get more than 15volts;
finally check if your pvdf membrane supports methanol. Some memebranes do not support methanol.
keep us informed
my buffers (found in maniatis):
running :
glycine 1,92M
SDS 1%
tris base 250mM
for one liter, it is 144.13g 10g and 30,28g respectively
transfert :
glycine 1,92M
tris base 250mM
for one liter, it is 144.13g and 30,28g respectively
If you want, you can add methanol :
10X transfert solution 100 ml
methanol 200ml
ddh2O qsp 1 liter.
Thank you for Ur suggetion
We tried without SDS in this case we are getting some time result and some time no transfer.
One more problem we are facing that, we having Biorad PVDF roll and when we are dipping this PVDF to transfer buffer (priviously dip in methanol for 10-15 sec) it get seperating in to two membrane (pouch like ).
Some time wrinkle coming in PVDF
Is this problem in PVDF or our transfer technique
Regards
Dhiraj
I think the membrane, and/or the transfer buffer, there is something int he transfer buffer that cause your membrane wrinkle, check the Catalogue of that membrane and see its composition.
for your transfer, I believe that 2 hours is too much. You can try to transfer at 20 volts for 20 minutes or 30 minutes, or do a wet transfer for 2 hours at 350mA.
for transfer buffer use the following
tris base
Glycine
DDH2O
dissolve and chill at 4C then before use add 20% methanol
good luck