I run the electrophoresis in a pre-cast 4-20% gel at 200 V for ~45 min, then i've tried to transfer them to nitrocellulose membrane in the following conditions (always in a cold, wet environment):
* Transfer for 30 min in 20% methanol buffer (Tris 25 mM, Gly 250 mM, SDS 1%, Methanol 20%, pH 8.3) = ZO-1 bands were blurry or not there
* Transfer for 1:15 in 5% methanol buffer (Tris 25mM, Glycine 192 mM and methanol 5%, no SDS, pH 8.3) = ZO-1 bands were OK, but CLD-5 didn't appear
Hi Blanca
I recommend you to use PVDF membranes in order to retain more protein. Also you should try to transfer in semi-dry conditions to search the 22 KDa protein.
I use the second buffer to run my electrophoresis but with SDS and add 10 % methanol for transfer, also with SDS. I transfer for 90 min at 320 mA with constant amperage, . I find ZO-1 and other protein of 22 kDa.
Finally, if you run your electrophoresis to fast and with low purity reagents the small proteins always are diffused.
Good luck
As simple as you standardize with low and high MW protein marker primarily in gel, then cut the gel @ 10-30 KDa, and same for 220KDa after transfer, fiinally separate membranes each and do further experiment as for your SOP's.
Use PVDF for better result
in another method stripping buffer might help for both protein identification.
Good luck
The easiest way is to use a gradient polyacrylamide gel electrophoresis like 4 % to 15 %. There are plenty of commercial gels.
- Badges
- Science method