semi-dry western conditions - (Mar/25/2004 )

hello,

Try reducing the time of transfer to approximately 10-15min at 100mA. Reason, protein is fairly small, so it will traverse the membrane if left to transfer for an extended period of time. best of luck .

Joshua

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hi
I had this problem before, then I used a PVDF (0.45umpore size)
I activate the membrane for 2minutes in 100% methanol, then soak the membrane in transfer buffer for 5-10 minutes, I transfer at 20Volts for 20 minutes, and I always get a good result. But wet transfer is the best specially for 2-D gels.

regards

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I'm looking for a 47kDa protein using the same method and it is working: run on a 10% gel at 100-150V, then blot using a semi dry run at 15V for 30mins., I use a PVDF membrane, soaked in meOH for 5 minutes, then equilibrised in transfer buffer for a few washes. renmember to soak everything, gels inc, in transfer buffer before blotting....

hope this helps!

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Semi-dry has been working perfectly for my proteins.
But I have heard complaints from colleagues next door about one of their proteins that does not transfer at all in semi-dry, yet transfers very well with conventional transfering system (the sandwich in a tank with ice block).
Have you tried with the conventional transfering system?

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first check your buffer system (prepare fresh) and if possible use on prestainedprotein marker.usually 35 volts for 25 min is more than enough for semi dry .make sure your whatman filter papers are equally placed in the sandwih.and all the papers should be in equal size .and finally prepare a fresh ponceus reagent .

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never regret keep going till you reach the required,

sudha

i did not come across any sort of trouble in transfering the proteins of low molecular wts approximately 6.5 kd to higher around 130 kd with the trans-blot semi-dry transfer cell apparatus of bio-rad from an 8 % gel on to pvdf , and the transfer is at 15 v for 30 min( i would like to check the same in 10 and 12 % gels ) . but the higher mol wt proteins (more than the mentioned above) were still in the gel revealed by coomassie.i think some more modefications are required for effective transfer of all the proteins.

all the best

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we usually use constant Amp 180mA (voltage no more than 25V) for 1hr. Also, adding methnol into transfering buffer probably will help bit.

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Hi all,

I am very confused by reading everyone's opinions about semi-dry techniques

I use 200V (or 0.05A) to transfer for around 75-80 minutes, and the transfer is not a 100% as i see my proteins
after coomassie staining.

I block in 5% milk for 1 hr, then primary overnight, 3 washes, follwed by a second blocking step with milk for 30 min, then secondary for 1 hr, followed by 4 washes.

I have had no luck using this technique to see my proteins which are in the range of 80 and 200kd.

Should I use a higher voltage during transfer??

Thanks

Pooja

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Hi
when you use the semi dry, the order of the transefer sandwich should be, wet filter paper, membrane, gel, filter paper.
then if you are using PVDF membrane you have to activate it with methanol, then soak it in buffer for few minutes. you also have to calibrate your gel in the buffer as well for few minutes. for higher molecular weight proteins, you don't expect to see all your proteins get transfered, maybe 50% or less will go on the membrane, I usualy use 20volts for 45 minutes. If you are still having trouble, try wet transfer, I get more than 90% transfere using biorad wet transfere apparatus. good luck

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