I am trying to do 2d gel electrophoresis for the whole protein of cell lysate. and using aceton precipitation I clean up my lysate. I have Urea, CHAPS and IPG buffer3-10 in my rehydration buffer. I am using 7cm, 3-10 nl strips. I always measure my protein amount using nano drop or bradford. It is a long time that I am doing this and I always try to be consistence in what I do and repeat everything the same. But so far I have not seen two nice gel in a row. It works one time and not the other time. And by saying not working I mean NOTHING in the gel. Can you think about anything that I am ignoring or not paying enough attention?
By the way, I am using silver staining with homemade reagents.
Thank you very much in advance.
E.g. temperature and pH have an essential influence of the outcome of 2DE-PAGE.
Protocol schould be followed as exact as possible, including same environmental conditions.
Andreas Eisenreich Thank you for your response. I will be more careful for these conditions.
Did you use Nanodrop and Bradford? I made the experience, that Nanodrop sometimes measures imaginary things, so I´d prefer to use Bradford.
The manufacturer of your strips should mention somewhere the maximum amount of liquid loaded onto a strip. Make sure not to exceed this volume.
I had always a very small amount of Bromophenol Blue in my rehydration buffer. During IEF the colour will disappear, if not, your IEF didn´t work.
Another thing is the rehydration time, was it always the same? And under the same conditions?
Uwe Wegner Thanks for your detailed answer. I used to measure with nanodrop I just started using bradford. I am loading 125ul on 7cm strip and my BPB disappears completely during IEF. and my rehydration time is always 20h RT.
Is there a certain time that proteins should wait in rehydration buffer before loading to strip? I mean can it be about incomplete reduction of proteins?
I don´t think that it is a problem with incubation time, reduction continues during rehydration.
A simple but fatal error might be, that you have forgotten SDS in your running buffer...
Did you try another staining method? Maybe there´s something wrong with your reagents. Coomassie is a quite easy method, just to check, if electrophoresis worked. And it´s sensitivity is not that bad as it was several years ago.
Uwe Wegner Actually I am suspected about staining part too. I have good result with 10ug protein loading but this could be not a accurate protein measurement since I did with nanodrop. I will go for higher protein amounts and if not working as you said I will go for Coomassie. I will let you know. Thank you very much.
Hi
What voltage do you work with? Is the voltage time applied to the device fluctuating or increasing? and are u sure about staining protocol? you can try SILVER-BLUE protocol as the simple and High quality way.
If you have checked the protocols, It's a good idea to change the staining scheme.
Mohammad Mohseni Thank you for your response.
Here is my protocol of focusing : 1st step and hold 300 V 30min
2nd Grad 1000 V 30 min
3rd Grad 5000 V 1:30
4th step and hold 5000 V 25 min
at the end of run what I have as total Vhr is not not constant for all runs. for example for a run that I had good result, it was 6949 Vhr and for the one not working it was, 6261 Vhr. I dont know if such difference between run is meaningful or not.
If the voltage does not increase, it indicates the presence of salt in your sample; Also, the presence of IPG Buffer in large quantities causes resistance to the current (A) in the strips. Please make sure the samples are free of salts.
I was involved with this problem last year; that was not spot in the gel, so I'll transfer any point that comes to my mind.
So, can you find any dark area on the gels؟ Not spots, only black or dark area or smear؟
Mohammad Mohseni Thank you very much for your help. I use acetone precipitation to get rid of salts and I have 0.5% IPG buffer. yes in some gels I have no spots but black area.
That sounds like a bad separation in both dimensions.
During IEF you should have a moist (not wet!) piece of paper between your strip and the electrodes. Do you use always the same size and the same amount of water?
This paper is important to remove remaining salt or cell debris and other unwanted things from your sample during the first steps of IEF.
Another reason for a black area can be, that you simply overloaded the strip. If the protein concentration is higher, than you measured, you might have more protein on the gel, and the spot don´t have enough space to be separated. Do you see one large area, or just a few small areas?
If it is one large area, check your protein concentrations.
For bacterial whole cell proteomes, I used strips with a pH gradient 4-7 for a better resolution in the first dimension, maybe you can try them.
- Badges
- Science topic
- Similar topics
- Chemistry
- General Biochemistry
- Proteomics