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Transfer problems-smeared blot
I am new in western blotting, and have been experiencing some difficulties in transferring. After I ran the gel, the ladders are straight and everything seems normal. However, everytime I use second time transfer buffer to transfer, my resulting blot shows that the ladder in the middle of the blot is always smeared and even disappears on the top. However, whenever I use first time transfer buffer, there are no smears.
My supervisor says they have been experiencing this problem whenever they use second time transfer buffer for over half a year now and would like me to find a solution. (Half a year ago, they never had problems when reusing the transfer buffer solutions even up to 4 times, but they dont know what caused this difference)
There could be something wrong with the transfer buffer recipe, or our techniques.
So here are some details:
10x Transfer buffer recipe (4L):
58.40g Glycine (I found online that usually you should add 57.6g of glycine for concentration of 14.4g/L, but I dont think such a small difference would cause the transferring problem)
-adjust pH to 8.3
-fill water to make 4L
-diluted to 1X transfer buffer by adding 100ml 10 X transfer buffer, 200ml methanol, 700ml ddH2O
-Before gel is done running, I soak the PVDF membrane in 100% methanol for about 10-15mins.
-I also soak the filter papers and sponges in transfer buffer taken from 4 degree celsuis for 10-15 mins, while rolling out bubbles with a 15ml conical tube.
-When gel is done running, i take it out and soak in transfer buffer for 5 to 10 mins.
-I assemble the sandwich, by placing sponge, 2x filter paper, the gel, membrane, 1 filter paper, then sponge.
-We use Biorad transfer tank, with an ice pack placed inside to prevent overheating.
-I usually transfer at 90V for 40 mins or 100v for 45 mins with transfer buffer taken from 4 degrees poured to cover the cassets holding the sandwich.
-When transfer is done, i can feel that the transfer buffer is slightly warm. However, when it was first time transfer buffer used, there is no smears on the blot. When it is second time transfer buffer, there is smears in the middle of the blot, but never the sides.
-also my supervisor has tried before to transfer in ice, but smears still resulted.
-she also tried to use a magnetic stir bar to distribute the cooling effect of the ice pack evenly inside the tank, but smears still results.
- I found online that it says "PVDF membranes require careful pre-treatment: soak it in methanol for 1-2 min. Incubate in ice cold transfer buffer for 5 minutes. The gel needs to equilibrate for 3-5 minutes in ice cold transfer buffer. Failure to do so will cause shrinking while transferring, and a distorted pattern of transfer.”
Could I be soaking the membrane in methanol for too long? (10-15mins) Could that cause the smears? Or maybe it's because I dont soak the membrane in transfer buffer? I am going to try following this instruction next time I transfer, but i doubt it will solve the problem, since they are just little adjustments.
-could it be the transfer buffer recipe, where my recipe has a bit more of glycine? (58.4 instead of 57.6g) But we still have a lot of 10x buffer left, and therefore wont be remaking it for awhile. And I dont think that little amountmakes a difference.
I am sorry for such a long post! But I am really new to western blotting, so I could need a lot of suggestions and help! Anything would be appreciated!
Re: Transfer problems-smeared blot
Ok, 10-15 minutes in pure methanol seems a very long time to soak in methanol. We do about 10 seconds followed by 10 minutes in transfer buffer. This wont be the problem though if your fresh transfer buffer is ok but reused isn't.
Are there any stages during the run or assembly where your buffer is exposed to air without a lid for long periods? Methanol with evaporate off the surface quite fast. You might want to try testing the pH of a batch of buffer after its been run once and twice to see if it changes significantly.
What is the volume of your transfer tank? High voltage can sometimes cause problems especially if the gel loses some of its rigidity.
|blot , problemssmeared , transfer|
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