After many time running IEF and PAGE, I never get spots throughout the gel. I am using 18 cm strips (pH 3-10) strips from BioRad and GE also. Is there any expert/person involved in this 2D suggest me any precaution or correct procedure for running 2D? Please share me your views!
Thanking you
Make sure you are following a working recipe exactly as written
try the following: Load 2 strips with about 1 ug of BSA each (slightly acidic protein) in IPF loading buffer (or equilibration buffer if you are doing rehydration-loading). Run both strips. Take one strip and stain with coomassie blue R250. Take the second strip and run the second dimension. Again, stain with coomassie. The sensitivity of coomassie is about 0.1 ug protein. This should tell you if you are having a problem with your sample entering the first dimension gel or transferring from the IPG strip to the SDS-PAGE gel in the second dimension.
Dear Yashvant
Any comments will be applicable after seeing your 2D maps. However, I had similar experience.
What is the brand of electrophoresis apparatus of the 1st and 2nd dimensions?
I think that your gel is become to hot and therefore, you can not find any spots. Please keep the gels cool.
If not, it is better to load more protein concentration.
It is better to run multi-steps IEF to prevent any shock to the protein (you can see one of my article which published in Electrophoresis 2014)
Also, check the salt concentration before electrophoresis.
If you can not find any spots with these suggestions, I recommend to cut the 18 cm strips to 2 pieces and the run in smaller trays and set of electrophoresis.
Dear Yashvant,
to give a helpful answer it is necessary to know more about your samples and buffer conditions. Mrs. Lamy is right, it could be a too low protein concentration. Fluorescence labeling can help there as it is much more sensitive as CBB (0,03ng). This would in general give you the opportunity to check the strips after IEF and the gels after running the 2nd dimension without staining, which is very handy for every 2D-PAGE.
If it is not the protein concentration it could be salt in your sample (the strip can start to burn with high amounts of salt), it could be precipitation while IEF, it could be problems with protein transfer from strip to gel. To exclude problems with the equipment you can ask another lab to analyze your samples with your SOP (we could do that). To exclude contaminations the 2D-cleanup kit from GE is very good or a simple TCA precipitation. Running a normal 1D gel with the clean samples before you start with the IEF should also be part of your SOP (again this is very fast and simple using fluorescent labeling) to check your samples before using the expensive strips.
Our 2D Coach might help further after sending some pictures of your gel and more information about your samples and SOPs.
Kind regards,
ristin
Hello All, Thank you very much for all your suggestions
Details of our experiment as:
Samples were solubilized in a buffer containing 8M urea, 2M thiourea, 4%CHAPS, protease inhibitor cocktail and in last 40mM DTT. 100microg protein sample was rehydrated on GE strips in rehydration buffer containing 2%CHAPS, Urea (8M), 2M thiourea, IPG buffer (0.5%), bromophenol blue and DTT for 18 hours at 20 degree temp. After rehydration, IEF wes done in GE instrument with program: Step 1: 200V for 1 hr, Step 2: 500V for 3 hr, Step 3: 1,000V for 4 hr, Step 4: 8,000V for 2 hr and Step 5: 8,000V for 13 hr.
Then equilibrate the strips in SDS-Equilibrium buffer containing DTT for 15 min and iodoacetamide for 15 min, then second dimension electrophoresis in sixDALT system of GE. Staining by silver staining.
Now, from all of you, wish to know/discuss the actual problem what I were facing since last month
Thank you in advance
Sorry I forgot in mentioning that I am working on Drosophila and I uses Eye imaginal discs for protein work.
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