I'm going to do western blot to detect a low abundance protein, PVDF membrane is my choice, but which transfer method is better?? and what are their limitation???
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#2
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Posted 16 February 2004 - 04:12 AM
Hi there!
We're using both methods in our lab, so maybe I can give you some advice, though i can only tell you the difference between biorad's tank blots and biometra's semy-dry...
Each method has it's own pros and cons.
Semidry blotting is faster and easier, and reqires a lot less buffer volumes than tank blotting. Small Proteins (<20kDa) are transferred with better reproducibilty. Big proteins, however, do not transfer well in semidry blotting, and blotting of those is random at best, i.e. in some semidry blots you get big proteins on the membrane, sometimes you don't (same conditions!).
Tank blots transfer high molecular weight proteins (>60kDa) better than semi-dry, but more often than not some small proteins are blotted trough the membrane and are therefor lost for detection.
For the proteins I work with (25-35kDa) both methods work equally well, but I personally prefer the semi-dry blot, since I don't like wading though knee-deep in blotting buffer and preparing fresh buffer every other day. But for detection of BIG proteins (130kDa) I change to tank blots....
Mike
We're using both methods in our lab, so maybe I can give you some advice, though i can only tell you the difference between biorad's tank blots and biometra's semy-dry...
Each method has it's own pros and cons.
Semidry blotting is faster and easier, and reqires a lot less buffer volumes than tank blotting. Small Proteins (<20kDa) are transferred with better reproducibilty. Big proteins, however, do not transfer well in semidry blotting, and blotting of those is random at best, i.e. in some semidry blots you get big proteins on the membrane, sometimes you don't (same conditions!).
Tank blots transfer high molecular weight proteins (>60kDa) better than semi-dry, but more often than not some small proteins are blotted trough the membrane and are therefor lost for detection.
For the proteins I work with (25-35kDa) both methods work equally well, but I personally prefer the semi-dry blot, since I don't like wading though knee-deep in blotting buffer and preparing fresh buffer every other day. But for detection of BIG proteins (130kDa) I change to tank blots....
Mike
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#3
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Posted 17 February 2004 - 02:22 AM
thanks Mike
I also have doubt on another issue. That's protein transfer through the membrane and reach filter paper...lost of target protein!!
Both semidry and tank blot encounter this problem??
I've experienced that large protein (the marker shpwed that) transfer through the membrane! Will tank blot also have this problem?? Also large protein???
I also have doubt on another issue. That's protein transfer through the membrane and reach filter paper...lost of target protein!!
Both semidry and tank blot encounter this problem??
I've experienced that large protein (the marker shpwed that) transfer through the membrane! Will tank blot also have this problem?? Also large protein???
#4
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Posted 17 February 2004 - 07:59 AM
Hi again!
Yes, blotting through the membrane can happen with both systems....
If that's your problem, try to shorten the blotting time and/or lower the used amperage/voltage... Do some test blots with your standard and observe when at which conditions the transfer for a given size of protein is best...
Mike
Yes, blotting through the membrane can happen with both systems....
If that's your problem, try to shorten the blotting time and/or lower the used amperage/voltage... Do some test blots with your standard and observe when at which conditions the transfer for a given size of protein is best...
Mike
--- He who finds typos may keep them! ---
#5
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Posted 24 August 2004 - 01:40 AM
do wet everytime, it might be messy and use loads of buffer but it almost always gives better transfer
#6
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Posted 15 September 2004 - 12:09 PM
Hi, I don't know if this is helpful but I also tried both semi-dry and wet transfers only because I wasn't getting a good transfer and because I had really crappy (only available) antibodies for detection. If you aren't getting a good wet transfer adjust your methanol from 200 ml/1000 ml to 150 ml/1000 ml without altering your glycine. That was the trick that allowed mt to get the best wet transfer and was superior to the semi-dry. Hopefully, I will complete the manuscript and get it published soon.
#7
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Posted 09 May 2012 - 05:08 AM
Well the statement that proteins go through PVDF membrane is largely not true the usual pore size is 0.22micrometer which is usually good enough to retain most of the proteins (we are not talking about small peptides).
A great devices to transfer protein is the Invitrogen iBlot mahine, transfer is done efficiently in 8mn and probing gives excellent results (we are using it daily)
A great devices to transfer protein is the Invitrogen iBlot mahine, transfer is done efficiently in 8mn and probing gives excellent results (we are using it daily)
#8
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Posted 09 May 2012 - 07:50 AM
pesji, on 09 May 2012 - 05:08 AM, said:
Well the statement that proteins go through PVDF membrane is largely not true the usual pore size is 0.22micrometer which is usually good enough to retain most of the proteins (we are not talking about small peptides).
assuming you are correct (i'm not sure you are, i see a lot of 0.45um pore size membranes used), in 2004 the usual pore size was 0.45um. and, there are other factors which may lead to blow through, even with 0.22um pores.
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genius does what it must
i do what i get paid to do
#9
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Posted 15 May 2012 - 06:36 AM
mdfenko, on 09 May 2012 - 07:50 AM, said:
pesji, on 09 May 2012 - 05:08 AM, said:
Well the statement that proteins go through PVDF membrane is largely not true the usual pore size is 0.22micrometer which is usually good enough to retain most of the proteins (we are not talking about small peptides).
assuming you are correct (i'm not sure you are, i see a lot of 0.45um pore size membranes used), in 2004 the usual pore size was 0.45um. and, there are other factors which may lead to blow through, even with 0.22um pores.
Never seen a protein going through our PVDF membrane so I don't say that it cannot happen but I guess we are running enough Wertern blot here to say that we havn't experienced it
