Semidry Transfer - (Feb/19/2009 )
Quick, simple question
I am now in a new lab which has never done western before
And in the past I did lots of western, but all wet blot
now the lab has a semidry blot, and I did the first transfer yesterday
Using Bio-RAD Transfer SD
2 minigels (invitrogen, 1mm) 15V constant voltage, 30mins (following Bio-RAD instruction)
I looked briefly, and the marker is on both the PVDF and the gel,
So I continue (or restart) the transfer at 15V, 30mins again
looks better, but I can still see the marker on the gel,
And from comassie blue staining, I can still see protein in the gel
In the past, when I do wet blot, it's always complete
So maybe I used a wrong condition?
What voltage and time you guys use?
It's been a while since I semi-dry transferred but there are several important aspects.
1. Transfer at constant AMPs. Not volts. Very important. I think I transferres at 200 miliAMPs.
2. Use either the thick blotting paper from Biorad or 4 thin Watmann squares on each side. \
3. Make sure everything is really well soaked in buffer. I pour a little extra on top.
4. Transfer for 1 hr. NOT 30 minutes.
5. Semi-dry isn't very efficient at transfering anything over 110 kDa.
mikew on Feb 19 2009, 11:33 AM said:
1. Transfer at constant AMPs. Not volts. Very important. I think I transferres at 200 miliAMPs.
2. Use either the thick blotting paper from Biorad or 4 thin Watmann squares on each side. \
3. Make sure everything is really well soaked in buffer. I pour a little extra on top.
4. Transfer for 1 hr. NOT 30 minutes.
5. Semi-dry isn't very efficient at transfering anything over 110 kDa.
Really helpful! Thanks
I'll try that
Agree with mikew.
We transfer at 40 mA for each minigel (i.e. 80 for two, and so on) for 1 h.
As for the transfer of large proteins, I found that reducing the % of the gel increases the transfer efficiency for those large proteins. For example, the ladder is still visible on the gel after transfer of a 10% gel, but not on a 6%.
madrius1 on Feb 19 2009, 12:51 PM said:
We transfer at 40 mA for each minigel (i.e. 80 for two, and so on) for 1 h.
As for the transfer of large proteins, I found that reducing the % of the gel increases the transfer efficiency for those large proteins. For example, the ladder is still visible on the gel after transfer of a 10% gel, but not on a 6%.
So let me report the outcome:
2minigel, I used constant current 200mA, transferred for 1hr
at the beginning, the voltage is 10V, and at the end, it became 41V
the transfer is mostly complete except things above 100kD (which I don't care)
so it basically works much better than constant voltage 15V (suggested by Bio-RAD!!!)