I tried to transfer today and found that the membrane (membrane 1) looked like this after ponceau S staining.
Two days ago, I transferred two membranes together in the same cassette and found this after developing (left and right blots). The left blot is good but the right one is like membrane 1.
What could be wrong.The problem is that how come same transfer conditions lead to one blot transferred correctly and other bad?
My transfer conditions:
1. constant current : 400 MA
2. Time (45-50 minutes)
3. Temperature 2-4 C. A magnetic stirrer was used to ensure constant agitation and temperature.
4. Buffer : 25 mM Tris, 192 mM Glycine, 20% (v/v) methanol, pH ~8.3.
Did you treat both blot exact same way, equilibrated membranes and removed bubbles. It is hard to understand what you mean by good in those images, but it looks like you have intense degradation in the first one
If small protein- 400 is quite high. You may reduce methanol to 10% and try overnight run at 20 to 25 V
Insufficiently close contact between the membrane and the gel is a likely problem. The sandwich should be under enough pressure to assure that the contact is very close and firm. You can add an extra layer of filter paper between the sponge and the gel or membrane (or both) as necessary to increase the pressure.
@ Adam: i had noticed that the cassette was unusually thinner than usual. Do you suggest adding one more sponge.
You can use another sponge if it will fit, or layers of filter paper. Make sure to squeeze out any air bubbles from between the gel and the nitrocellulose/nylon/PVDF filter.
I used
1. Another sponge and it worked
2. I used more filter papers and it worked.
Thanks.
You must fixed your cassette in a way that there's no any space in the end and remove all the bubbles carefully.
- Badges
- Science method