6 replies to this topic

[toptea]

Hi all,
I am optimizing my western blot from the last month. I am using nitrocellulose membrane and my target protein is around 45-55 KDa. Using SDS-PAGE Marker as well as Western C marker for detection by chemiluminescence, transferring by Bio Rad's turbo blot and imaging by versa doc, following the protocols.

Previously used Standard transfer for 30 min but can't find any band, switched to HMW for 10 min and got the bands for protein of my interest. Problem is I can't see the marker on the blot, If I reduce the number of washes from 6 to 3 then I can see few bands of marker (not all) with lot of background. In order to get neat picture, if I increase the number of washes, then can't see any band of the marker,
I was guessing, it may be due to insufficient transfer of proteins and marker, not sure...
Please help me, it seems like if I get the protein band, then no marker and vice versa.

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[bob1]

If you could provide a few more details as to how you are doing the transfers, this will help us troubleshoot. The "standard" protocols are as varied as the number of proteins that people use.

[toptea]

Thanks bob1 for your kind reply. I am making my own gels (10% resolving and 5% stacking), buffers and transfer packs. I am using the transfer method given in the list (built in transfer protocols) of Turbo Trans-Blot (Bio Rad's). I contacted the Bio Rad's technical team as well to get advice on this and they suggested to use High Molecular Weight (HMW) for 10 minutes transfer instead of Standard transfer for 30 minutes as it may have blown the proteins out of membrane. the transfer buffer I am using, contains 3.03 g of Tris-Base, 14.4 g of glycine and 100 mL of methanol in 1L. Trying to take care of all measures like use of fresh buffers etc.

[mdfenko]

what is the pore size of the membrane? we routinely use 0.2um pore nc for hmw and lmw blots.

please give us the protocol that you're using. the hmw protocols that i'm familiar with include 0.05% sds and 20% methanol in the transfer buffer.

[toptea]

Hi mdfenko, I am using 0.45 um pore size nitrocellulose membrane and used the protocol given on this forum (EnCor Biotechnology, http://www.encorbio....ls/blotting.htm) prepared the gels using protocol from Sambrooks book. Initially I used the standard transfer method as my protein weight range is 45-55 KDa, Problem was I couldn't see the marker on the blot and contacted Bio Rad's team and they suggested to use HMW transfer method (1.3 A; up to 25V)

[mdfenko]

you're using the trans-blot turbo system and program but you're referencing the procedure from encor biotech.

the procedures are not the same.

are you using the turbo transfer packs or home made?

either way, you should be following the procedure given with the instrument.

different systems have different requirements. we use a semi-dry system which uses 3 buffers and cannot be run for more than 1 hour (nor is it necessary) due to the composition of the electrodes.

if you followed the encor procedure then your buffer may not have contained enough methanol to strip the sds from the proteins. they may have passed right through the membrane.

i recommend that you follow the procedures given with the apparatus and i highly recommend that you use 0.2um pore size (this is also the pore size of the membrane with the transfer packs).

[toptea]

Thanks for your reply n recommendation, yes with the trans blot turbo, ready made transfer packs are recommended but I am making my own transfer packs. will update about the result after changing the transfer conditions. Regards