poor electrophoretic protein transfer - (Jun/18/2008 )
Hi everybody!
Recently I've found some problems with my electrophoretic transfer on nitrocellulose membrane. I use the same method from years and I've never had problem with it, so I think something get broken but I really tested everything! I usually make overnight transfer at +4°C and I've tried to increase power up to 26V. Transfer buffer is always the same (for 1 liter: 14.44g glycine, 3g Tris, 0.2g SDS and 20% MEOH). At the end of the transfer I always detect by gel stainig most of the proteins (if not all) still on the gel. Can anyone help me?
Recently I've found some problems with my electrophoretic transfer on nitrocellulose membrane. I use the same method from years and I've never had problem with it, so I think something get broken but I really tested everything! I usually make overnight transfer at +4°C and I've tried to increase power up to 26V. Transfer buffer is always the same (for 1 liter: 14.44g glycine, 3g Tris, 0.2g SDS and 20% MEOH). At the end of the transfer I always detect by gel stainig most of the proteins (if not all) still on the gel. Can anyone help me?
I've always had proteins remaining on my gel when i stain it after transfer - unless you have a before and after comparison its hard to tell if the transfer worked. A better method is probably to stain your membrane with Ponceau's to see if the proteins transferred. If you've done this and still can't see anything, then maybe you should check your transfer apparatus to make sure that the electrodes are in proper working order. Also, i've heard that if your methanol is old that can affect transfer. Also can you see that your protein markers have transferred?
I've always had proteins remaining on my gel when i stain it after transfer - unless you have a before and after comparison its hard to tell if the transfer worked. A better method is probably to stain your membrane with Ponceau's to see if the proteins transferred. If you've done this and still can't see anything, then maybe you should check your transfer apparatus to make sure that the electrodes are in proper working order. Also, i've heard that if your methanol is old that can affect transfer. Also can you see that your protein markers have transferred?
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I usually run two identical gels, then one is stained, the other transferred. I use ponceau (solution by SIGMA) too, and I see only few mild protein bands transferred. But problem is that transfer in not homogeneus, some areas transferred, others not.
Apparently the power supply works properly, if I set 30V costant, it shows about 75-80mA that should be correct. Moreover I use the same power supply for SDS-PAGE and I don't have problem with it... by now!
How can I check my transfer apparatus and the electrodes?
Are you doing wet transfer or dry transfer? By your description that some areas are transferred - is it one particular area of the gel, or random? Do your proteins look like they're swirling? I had some issues in the past with my membranes not making good contact with the gel and fixed the problem by using an additional pad - I use wet transfer. Perhaps your problem is similar and increasing the pressure of the membrane against the gel will help.
I use wet transfer, too. Random areas are transferred, and I've thought that it could be due to bad contact between gel and membrane. So I added two more filter papers, but it didn't work. Now I will try adding an additional pad, as you suggested. Thank you!
I use wet transfer, too. Random areas are transferred, and I've thought that it could be due to bad contact between gel and membrane. So I added two more filter papers, but it didn't work. Now I will try adding an additional pad, as you suggested. Thank you!
I really didn't realize what a difference the pads can make until yesterday. Ours were quite old, and as I told you I started adding a third one to my transfers because I was having problems. Well, yesterday I noticed that my boss had bought some new ones and so I tried them on my western. I couldn't believe how thick they were compared to the old ones! I could barely close the sandwich! So if your pads are pretty old or thin, and adding an extra helps, then I would suggest that you go ahead and just buy new ones.
Good luck to you!
smu
