I could not transfer high m.wt protein to NC membrance for western blotting. I tried the following ways
1. Wet transfer with transfer buffer + 0.1% SDS
2. Wet transfer with transfer buffer +0.1% SDS and 10% methanol
3. Wet transfer with transfer buffer +10% methanol
4. I tried running in 15v, 30V, 40v, 75V and 100.for different time periods.
All these methods did not work.
Let me know the best way.
Hello Divya,
The heaviest protein that I have analyzed by WB is a 210 kDa protein. But I detected without problems, my protocol was:
Wet transfer with transfer buffer: Tris 25 mM, Glycine 192 mM, Methanol 20%.
The blotting could be perform in two different ways:
90-100 V, 2h 30 min, 4ºC
30 V, Overnight, 4ºC
Personally I preffer the second one, because you don't heat up the transfer buffer, and in general you have a complete transference to the membrane. Do not use SDS. This protocol is optimized for a NC membranes. Do not forgot to check if the transference worked, i.e staining the membrane with Ponceau red (it can be removed later with TBS-Tween). And if your protein is a membrane protein, sometimes microwaving the membrane rinsed in PBS is usefull to increase the detection (check the bibliography).
I hope this will help you
Hello DIvya,
Also, check the pI of your protein. If the pI is near the pH of your transfer buffer (8.3), you may need to try another buffer (such as CAPS/MeOH pH 11)
Presence of SDS, Methanol, Voltage/power and % of gel used effects the transfer of the protein to membrane. Please try to use lesser % gel. For more than 200kD less than =
Use 0.05% tween 20 in your transfer buffer. It has been used to successfully transfer nearly 300 kda proteins in our lab.
i got good result for m-TOR which has molecular weight of 289. we didn't used SDS in transfer buffer but just used in running buffer. generally we do transfer by adding 200ml methanol per one liter of trans fer buffer. (for one liter we add 14gr glycine+ 3gr tris base+ add double or triple distilled water to get the final volume of 800ml + 200ml methanol). i personally used 10% gel .if you want to use you can use 8% also. we did transfer at 35V and the temperature is 4ºC over night. we used PVDF membrane. after transfer you do the next procedure or you can also check with ponceau. doing transfer at higher voltages and less time may cause improper tranfer and heating.
not only transfer every step(running, transfer, blocking , 1º -Ab, 2º-Ab treatment washings, final substrate addition) is important and as of i know its better to use CST-Abs please check the literature and google images for getting information on mlot pattern.
loading dye or lamelli buffer proper preparation also important.
running buffer= 14gr glycine+3gr tris base+water
transfer buffer=14gr glycine+ 3gr tris base+ 200ml methanol+water
try with PVDF membrane and soak the membrane in methanol for ~5minutes.
PBST= 1X PBS one liter+ 250 micro liter tween.
blocking= 5gr milk powder + PBST to make final volume 100ml.
5% milk is better than BSA for blocking as BSA is giving background. prepare Ab solutions and blocking solutions in PBST.
i think it works. all the best.
if you have any doubts please contact [email protected]
I would add just one point to the protocols Sudhakar just described. After the milk powder is completely dissolved in either PBST or TBST, check the pH. You may find that the milk powder has reduced the pH by as much as 0.5 units. (At least, Carnation Skimmed Milk Powder has that effect on our TBST).
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