please help me by providing the protocol for entire PLANT proteins
Dear Jimmy
I had this headache once when I started doing this nice and exciting procedure. My first run was not so good. I had a few spots and many streaks on the 2-D gel. I thought that was fine, I will repeat the whole thing with some modifications. I re-extracted my proteins with more care, made sure there was no salts in them, used fine chamicals, sonicated the proteins to make sure they properly solubilized after removing the pigments, ran the isoelectric focusing for sufficient time and at adequately high voltage using good equipment from the same company that supplied the IPG strips. My heart was actaully beating so hard that I felt it was going to jump out. In the end of the procedure, I got nothing. The gel was even worth than in the first time. From this point on, I kept repeating the whole stuff for about 2 month, about 4 times a week with no thing at all. Every time I got a bad gel, I got more desperate to get it right. I ordered a full box of IPG strips and after a large number of repeats it ran out. I then talked to my supervisor and told him I going to quit because I have spent a lot of money on this and I am not getting anything productive but he was supportive and asked me to order a NEW box of strips. I did and repeated the procedure. On that day (a saturday) I was sure that nothing is going happen. BUT for my surprise, I got the best 2-D gel I have ever had in my life!!!
The problem was in the old aging IPG strips!
I wanted to till you this story because I know how you feel. 2-D electrophoresis is a long fragile procedure. I mean by fragile that any fault in a single step or in a single reagent you use can ruin the whole thing.
What you need to do is to revise the reagents you are using and then you may refer to this webpage: http://www.wzw.tum.de/proteomik/fileadmin/documents/2D_Manual-Gorg_2007.pdf
Best wishes, and sorry for the long answer.
Method of protein lysate preparation play important role in work of proteomics. Once I extracted protein and i did not get any spots in my 2D gel and when I changed the protocol for protein extraction I got alot of spots. please try to look for suitable protocol for cell lysate preparation. if you need noe for bacteria I can send you the fast and easy protocol
In my experience, if your stain is not recently purchased, it might have lost a big portion of its effect. 2D-SDS Gel electrophoresis can be a big hassle, especially when trying to match several replicates or observe slight differences in protein expression between strains. Complete accuracy in recreating the procedure is important. Try checking that your reagents are all fairly new, since they don't last as long as they should with 100% efficacy. The other reason you may not get detectable spots is if your total protein input is too low, i.e. the proteins expressed in smaller quantities need to be taken into account; if you want to observe these spots, you may need to add more ug of extracted protein to begin with.
- Badges
- Science topic
- Similar topics
- Biological Science
- Botany
- Plant Biology
- Plant Biotechnology