Hi, Thanks for looking at my message.
I got a problem about my western blot: My protein(called N300) is small(10
kDa calculated, 13 kDa and 20 kDa on 15% SDS-PAGE) and I expressed it as a
GST fusion protein. After harvesting the GST-N300 fusion protein, I cleave
it with thrombin and run 15% SDS-PAGE to separate GST protein, N300 protein
and uncleaved GST-N300 fusion protein. Then I transfer the whole lane from
SDS-PAGE gel onto membrane( nitrocellulose or PVDF). After transfer, I block
the membrane with 10% milk in TTBS-> wash with TTBS -> incubate with first
antibody(anti-N300) in TTBS -> wash with TTBS -> incubate with second
antibody ->wash with TTBS -> ECL reaction -> expose X-ray film.
After the whole western procedure, I stained the membrane and found N300
band disappeared totally. No band showed on film corresponding to N300 protein.
I did some trouble shooting:
1. Transfer is successful(After transfer, I stained membrane and saw very
good bands for N300 protein).
2. Use of Tween-20 in Western blot wash off N300 from membrane strongly(both
bands were washed off in 0.1% Tween-20 TBS in 15 minutes). But shaking in
TBS overnight did not make N300 bands fall off from membrane.
3. UV corsslinked NC membrane cannot keep N300 on membrane for Western.
4. Both 0.2 um and 0.45 um pore size nitrocellulose and PVDF membrane do
notwork well to keep N300 on membrane for Western.
5. Not use Tween-20 and shorten antibody incubation time to 30 minutes for
each antibody can keep up to 10% N300 band left on membrane, but the Western
film showed strong background and make the true N300 band on film very hard
to see.
6. I use the recombinant protein, therefore the amount of protein N300 is
not a problem.
Now I am going to try using gelatin or BSA to block membrane.
If you happen to know or get some experience about Western blot of very
small protein(such as 10 kDa), would you give me some suggestion?
My email address: huangz at citrus.ucr.edu
I shall very much appreciate your assistance.
John Huang on 7/16/96
