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uneven transfer of western blot

uneven transfer of western blot - Protocols and Methods Forum

uneven transfer of western blot - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 08-21-2003, 05:36 PM
EK
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Default uneven transfer of western blot




""Jayakumar, R"" <[Only registered users see links. ]> wrote in message
news:97101976F8A044468CA74FE11883B90E02048906@VIST A.roswellpark.org...
hi..
I use western blots (Mini-transblot aparatus of BioRad) to detect a
mitochondrial membrane protein and have to load upto 20 ug of protein per
lane on a mini-protean II mini gel (15%). I have 10 lanes in each gel.
Immunoprecipitation is out of question for several reasons. I use transfer
conditions of 90 V at 1 1/2 hours (and cooled with a ice pack changed every
45 min) for transfering my proteins onto PVDF Immobilon P membranes
(Millipore).
STaining the transferred gels show large amounts of protein still left on
the gel. Blots show uneven band intensities indicating uneven protein
transfer rates across the blot (more towards the outside and less towards
the inside). ACtually I am not that much concerned about complete transfer
of proteins, as much as I am about the uniformity of transfer (atleast same
rates) across the membrane. Actually I get complete transfer of proteins
when loadedin lower concentrations and hence my other proteins dont have any
problem. Any ideas as to what is going wrong.
By the way, I do wet the membrane in 100 % MeOH and then in transfer
buffer before using it for transfer. I use the Tris Glycine system with 20
% methanol and no SDS for transfer.
Any advice would be gratefully acknowledged.
Jai

---

From what I remember discussed before, 15% or lower MeOH, as well as
10-15-min pre-incubation of the gel in the transfer buffer should help. I
would even suggest pre-incubating the gel in the transfer buffer without
MeOH first (~5 min?), and then in buffer with 15% MeOH.
Emir.


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