2D-electrophoresis - “hollow” effect and streaking problems (Nov/11/2008 )
Hello everyone !
I have just started doing 2D-electrophoresis and need some help.
Does anyone have experience in the field? I have done a few runs with relative success, but have some questions…
I’m setting up the system with the idea of make a comparison between protein profiles from fibroblastic cells cultured in different conditions (control vs treatment) and detect spots with different intensity (proteins differentially expressed).
I run the 1st dimension on IPG strip pH 4-7. Rehydration loading overnight, aprox 70 mg protein, standard buffers (home made), IEF: 500V 1h - 1,000V 1h – 8,000V 2h. DTE and iodoacetamide equilibration prior to 2nd dimension. Silver staining using Silver Quest kit (Invitrogen).
I tried to attach an image of my last gel, but I couldn’t upload it.
1. Many spots shows the “doughnut” or “hollow” effect, which lost their central quantity... this is going to be a problem for the quantification !
2. In the lower half of the gel, the spots look pretty good, but in the upper part (high MW proteins) there is lot of streaking…
If anyone has a suggestion, references recommendation or any ideas about this it would help me a lot!
Thanks !!
Contact Dr. Soni via kas364@msstate.edu, who has lots of experience in 2-D gel electrophresis.
What you are seeing is quite typical.
Streaks are often caused by something called 'overfocussing', where the IEF gradient gradually breaks down and you get some drift of proteins because the gradient isn't uniform. Try doing your IEF for less time. (Google for a more detailed explanation and troubleshooting.)
Negative bands in silver staining are also quite typical. Various explanations for this mechanism are:
-protein is overabundant (although I'm not sure how this would affect the reduction of ionic silver).
-nature of the protein is such that Ag+ ions are not reduced, due to lack of sulfhydryl or carboxyl groups.
You can also find quite a lot more about negative staining by doing an internet search.
If you look at published 2D-gels, pretty much all have some streaking and negative staining, so don't feel as though you have atypical results or that you're doing something wrong.
Ginger
Streaks are often caused by something called 'overfocussing', where the IEF gradient gradually breaks down and you get some drift of proteins because the gradient isn't uniform. Try doing your IEF for less time. (Google for a more detailed explanation and troubleshooting.)
Negative bands in silver staining are also quite typical. Various explanations for this mechanism are:
-protein is overabundant (although I'm not sure how this would affect the reduction of ionic silver).
-nature of the protein is such that Ag+ ions are not reduced, due to lack of sulfhydryl or carboxyl groups.
You can also find quite a lot more about negative staining by doing an internet search.
If you look at published 2D-gels, pretty much all have some streaking and negative staining, so don't feel as though you have atypical results or that you're doing something wrong.
Ginger
The hollowing out of bands with silver staining is because of overloading, as ginger said. As far as I can recall, it is actually the deposition of a thin layer of metallic silver, which is highly reflective. If you were to use less protein, your very strong spots should come good again, but at the loss of the lowest-concentration spots. If your densitometer is good, though, you'll still be able to get some data on those weak spots, if that's what you're after.
Thanks a lot guys for your help !! and sorry for the delay in replying (I just had an accident...)
I have just started doing 2D-electrophoresis and need some help.
Does anyone have experience in the field? I have done a few runs with relative success, but have some questions…
I’m setting up the system with the idea of make a comparison between protein profiles from fibroblastic cells cultured in different conditions (control vs treatment) and detect spots with different intensity (proteins differentially expressed).
I run the 1st dimension on IPG strip pH 4-7. Rehydration loading overnight, aprox 70 mg protein, standard buffers (home made), IEF: 500V 1h - 1,000V 1h – 8,000V 2h. DTE and iodoacetamide equilibration prior to 2nd dimension. Silver staining using Silver Quest kit (Invitrogen).
I tried to attach an image of my last gel, but I couldn’t upload it.
1. Many spots shows the “doughnut” or “hollow” effect, which lost their central quantity... this is going to be a problem for the quantification !
2. In the lower half of the gel, the spots look pretty good, but in the upper part (high MW proteins) there is lot of streaking…
If anyone has a suggestion, references recommendation or any ideas about this it would help me a lot!
Thanks !!
Relative sucess? How you do it?
I have just start with 2D either and all experiences till now were ruined on the second dimension running... I don't know why but i simply can't put the strip on SDS gel and make the proteins in acrilamide without diffuse...
I believe we need to replace our old electrophoresis system to a new one but that's not possibl€ 
So I will keep tryng if someone have any sugestion I appreciate thanks in advance
I don't know what is going on with your second dimension, but I don't think the electrophoretic system is the problem. I'm using a prety old and very simple equipment (from R. Shadel inc.) and it works pretty good. If you want to check it, here is the link http://www.shadel.com/discovery.html
How do you know there is a diffusion problem and not something else?
I have no much expereince, but if you want my protocols to take a look, just let me know.
How do you know there is a diffusion problem and not something else?
I have no much expereince, but if you want my protocols to take a look, just let me know.
Dear all
a simple talk during the lunch break solved my problem... I wasn't doing it right and a collegue from the next door lab "show me the light" i was putting agarose in a wrong way and now i already got beautiful dots (they are so pretty
)thanks again