I am facing primarily 2 issues with Western Blot. Firstly, during the transfer process, lot of heat is getting generated that results in boiling I do put ice pack during the process, but lately the heat is very high. I run at 100V for 1 hour.
Secondly, my nitrocellulose membrane is turning pink after the transfer. I have no clue what might be the case.
Colina, I would check the electrodes to make sure there isn't any corrosion on them. What kind of transfer buffer are you using? If the salt concentration is off that may also cause an increase in temp. We run our wet transfers in the cold room and I normally set it at constant amps (200 mAmps for 2 hrs, but this various depending on what protein we're trying to get).
It might be due to the heat that is causing your membrane to change color, especially if you're using some sort of colored ladder/dye when running your samples but I'm just guessing on that!
Thanks David. I checked the electrodes. They look fine to me. I use 1X Tris-Glycine with 20% methanol in it. I too think its the heat that is causing color change, but I am not sure suddenly how this heat is building up.
Hi, Colina, you can use less voltage (20-25 V) and perform transfer overnight. I do not like high voltage becaus eoif the heating. In addition, in the case of high voltage you can easy "overtransfer" protein as David already mention: transfer condition strongly depoendent from protein soze.
You buffer is Ok, try overnight transfer in the cold room. You can insert your transfer chamber in ice bath.
I hope this will help!
Hello Colina,
The composition of the transfer buffer can greatly influence the temperature of the buffer while heating up. If you are diluting the transfer buffer from an existing stock, make sure that you dont add more of the stock than what you need. A slight increase in the aliquot size from the stock can translate into a higher ionic concentration in the final buffer. Ive had similar occurrences happen while running SDS PAGE and using adequate stock amount does help, the reason being an increase in the conductance in the buffer due to an increase in the molar concentrations of ions, that may contribute to the buffer heating up. Also have you tried using PVDF membranes rather than just nitrocellulose? PVDF can be better at times!
Best,
Ashutosh
AT the very beginning of an transfer from the SDS gel to the membrane an isotachophoresis is taking place. This is due to the pH differences between the gel and the transfer buffer, which is needed.
This created a lot of resistance and this a lot of heat. Therefore I prefer a wet / tank blot. here you can cool your cassete down (to 15 °C, below SDS will crytallize). Take your trime, be patient. Tank blots can run overnight. Constant temperatures helps you to keep the gel in a constant size, this avoid double bands, ... and it's much mor sensitive.
The semidry procedures are mostle not better than diffusion blots which can be driven by salt gradients.
The pink issue: try another supplier or membrane.
try to use in the transfer buffer preparation: glycine..tris base ..distelled water and methanol...
the membrane should be PVDF membrane it will work well
we routinely carry out transfers on nc membrane at 50 v, 150 ma for a 7x5cm, 0.75 mm gel. transfer takes approximately 1 h. works absolutely fine, with no cooling, but the assembly placed on a magnetic stirrer for contined stirring/dissipation of heat.
i agreeew ith vivek while transfering put it 100v for 1hr and 10 mins with a small magnetic stirrer inside the apparatus.
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