help optimize an western blot for 6kd protein - western blot tranfer problem (Mar/10/2008 )
QUOTE (Flour Power @ Mar 18 2008, 06:59 AM)
Hiya,
I also regularly do western blots for a 12kDa protein, and i have never once had any trouble. i make 12% tricine gels (although i am sure you could go ab bit higher than this for a 6kDa).
here is my transfer buffer for the western blotting:
39mM Glycine, 48mM Tris, 0.037% (w/v) SDS, 20% (v/v) Methanol, pH8.3 with HCl.
for a standard thickness mini gel (0.75mm) i blot for 18 mins at 13v on a biorad semi-dry.
my gels andf westerns are always perfect!
I also regularly do western blots for a 12kDa protein, and i have never once had any trouble. i make 12% tricine gels (although i am sure you could go ab bit higher than this for a 6kDa).
here is my transfer buffer for the western blotting:
39mM Glycine, 48mM Tris, 0.037% (w/v) SDS, 20% (v/v) Methanol, pH8.3 with HCl.
for a standard thickness mini gel (0.75mm) i blot for 18 mins at 13v on a biorad semi-dry.
my gels andf westerns are always perfect!
i know its quite hard to make tricine gels in my lab cause we dont have the degassing apparatus, thats why i use the tris, glycine gels, and we do wet transfers,s o can i still use the same buffer and time you suggested?
-- I wonder if you can cast the trice gels as glycine gels .. can you ? plz let me know
-mama wa nyumbani-
QUOTE (Flour Power @ Mar 18 2008, 06:59 AM)
Hiya,
I also regularly do western blots for a 12kDa protein, and i have never once had any trouble. i make 12% tricine gels (although i am sure you could go ab bit higher than this for a 6kDa).
here is my transfer buffer for the western blotting:
39mM Glycine, 48mM Tris, 0.037% (w/v) SDS, 20% (v/v) Methanol, pH8.3 with HCl.
for a standard thickness mini gel (0.75mm) i blot for 18 mins at 13v on a biorad semi-dry.
my gels andf westerns are always perfect!
I also regularly do western blots for a 12kDa protein, and i have never once had any trouble. i make 12% tricine gels (although i am sure you could go ab bit higher than this for a 6kDa).
here is my transfer buffer for the western blotting:
39mM Glycine, 48mM Tris, 0.037% (w/v) SDS, 20% (v/v) Methanol, pH8.3 with HCl.
for a standard thickness mini gel (0.75mm) i blot for 18 mins at 13v on a biorad semi-dry.
my gels andf westerns are always perfect!
flour power,
do you mind telling me what the pI of your protein is? Mine is quite high - 11. I was thinking of trying your transfer buffer but I don't know if you have this optimized for a different pI.
Thanks,
smu
-smu2-
QUOTE (smu2 @ Mar 19 2008, 05:17 PM)
flour power,
do you mind telling me what the pI of your protein is? Mine is quite high - 11. I was thinking of trying your transfer buffer but I don't know if you have this optimized for a different pI.
Thanks,
smu
do you mind telling me what the pI of your protein is? Mine is quite high - 11. I was thinking of trying your transfer buffer but I don't know if you have this optimized for a different pI.
Thanks,
smu
pI should not matter when you have sds treated protein. there will still be a net negative charge on the protein.
-mdfenko-
QUOTE (mama wa nyumbani @ Mar 19 2008, 02:45 PM)
i know its quite hard to make tricine gels in my lab cause we dont have the degassing apparatus, thats why i use the tris, glycine gels, and we do wet transfers,s o can i still use the same buffer and time you suggested?
-- I wonder if you can cast the trice gels as glycine gels .. can you ? plz let me know
-- I wonder if you can cast the trice gels as glycine gels .. can you ? plz let me know
you should be able to prepare tricine gels the same way you prepare glycine gels.
when you transfer small proteins and peptides you should use smaller pore membrane (ie 0.2um or smaller).
-mdfenko-
QUOTE (mdfenko @ Mar 24 2008, 07:59 AM)
QUOTE (smu2 @ Mar 19 2008, 05:17 PM)
flour power,
do you mind telling me what the pI of your protein is? Mine is quite high - 11. I was thinking of trying your transfer buffer but I don't know if you have this optimized for a different pI.
Thanks,
smu
do you mind telling me what the pI of your protein is? Mine is quite high - 11. I was thinking of trying your transfer buffer but I don't know if you have this optimized for a different pI.
Thanks,
smu
pI should not matter when you have sds treated protein. there will still be a net negative charge on the protein.
yes, but then why are there so many protocols that suggest using high pH transfer buffer for proteins that have high pI? I've seen many protocols which say to use CAPS buffer for low molecular weight, high pI proteins, although admittedly none of them really explain why. I tried this by the way, and the transfer was really great. The problem though, was that my antibody is weak and I have to use a femto ECL kit to do the western. When I did this, I got a reverse image - all of the transferred proteins were a white smear and the background was black. i tried to go to the regular pico ECL kit, but then I saw nothing. Thought that I would try the transfer buffer that four power recommended.
-smu2-
QUOTE (smu2 @ Mar 24 2008, 12:18 PM)
yes, but then why are there so many protocols that suggest using high pH transfer buffer for proteins that have high pI? I've seen many protocols which say to use CAPS buffer for low molecular weight, high pI proteins, although admittedly none of them really explain why. I tried this by the way, and the transfer was really great. The problem though, was that my antibody is weak and I have to use a femto ECL kit to do the western. When I did this, I got a reverse image - all of the transferred proteins were a white smear and the background was black. i tried to go to the regular pico ECL kit, but then I saw nothing. Thought that I would try the transfer buffer that flour power recommended.
the methanol in the transfer buffer strips the sds from the protein but if you add sds to the transfer buffer it should be of little to no consequence (although, sds interferes with the binding to the membrane).
otherwise, maintaining high pH should ensure that the protein will be negatively charged even if the sds is removed.
go ahead and try flour power's buffer.
-mdfenko-
QUOTE (mdfenko @ Mar 24 2008, 08:26 AM)
QUOTE (smu2 @ Mar 24 2008, 12:18 PM)
yes, but then why are there so many protocols that suggest using high pH transfer buffer for proteins that have high pI? I've seen many protocols which say to use CAPS buffer for low molecular weight, high pI proteins, although admittedly none of them really explain why. I tried this by the way, and the transfer was really great. The problem though, was that my antibody is weak and I have to use a femto ECL kit to do the western. When I did this, I got a reverse image - all of the transferred proteins were a white smear and the background was black. i tried to go to the regular pico ECL kit, but then I saw nothing. Thought that I would try the transfer buffer that flour power recommended.
the methanol in the transfer buffer strips the sds from the protein but if you add sds to the transfer buffer it should be of little to no consequence (although, sds interferes with the binding to the membrane).
otherwise, maintaining high pH should ensure that the protein will be negatively charged even if the sds is removed.
go ahead and try flour power's buffer.
Thank you for this explanation. I've been leaving out the SDS in my transfer buffer for the reason you mention. I'll try four power's buffer that has the SDS in it.
-smu2-