To improve resolution or less streaking on 2D gel, you should remove salt, detergent, phenolic compound, lipid and ionic contamination. Sample preparation has an important role in  high resolution of 2D gel. You  should choose very strong protein extraction buffer( depend on your  protein sample) to prevent protein aggregation or loss of solubility. Another important factor is PRTEIN IEF machine which should be set up in a proper  running program (voltage and hour). In summary,  horizontal spot streaking are caused by focusing time not optimized and unsoluble proteins.

Good Luck,

Nazila Nazemof, PhD

Proteomics researcher

As Nazila mentioned, factors such as salt, phenolic compound, lipid and other ionic contaminations may affect the focusing. Based on your 2D gel, it seems like the horizontal lines might be from your protein marker. The protein spots were not seriously horizontal streaked. In fact, there is some "wave" at the middle region which probably due to improper gel polymerization. It is good to cast the gel one day before and leave it overnight at chiller. Also, you may consider to load more proteins/increase the protein loading.

@Nazila Nazemof : Thanks for the suggestion. It would be of great help to me if you can suggest me the volt hour setting that I should give to a 7cm strip. What is the minimum and maximum?? Also if you have an idea on how to set the initial parameters please provide me with those. Infact we have removed the detergents,salts,lipids etc by methanol: chloroform precipitation. We have tried TCA/Acetone also, but since the yield was more on methanol:chloroform ,we thought of going with that. Interestingly the current in the IPG was showing only 35uA even-though we have set the maximun current limit/gel as 50uA, which might me a good indication that the sample is free of contaminants, I hope so. what are your view points on this aspect(sample preparation)???

Once again thank you very much

@Boon Chin Tan: We don't think like this is a problem arise due to the protein marker loaded. We suspect only problem in the first dimension because as far as i know, improper polymerization of the gel/ problem with the second dimension results only in vertical streaking. But will look in to this possibility also. Do you have any suggestions to provide regarding the voltage/parameters that I should provide in the IEF cell during first dimension?

Thank you very much,

Dear MDV,

I have different view from above researcher. Your gel is nicely focused as it is clear by the spot morphology.  The IPG strip which is not properly focused show spot with comet morphology having two tails at both ends. The second view regarding gel polymerization is also not acceptable as your marker showed proper separation. The problem in your gel is nucleic acid as you have used methanol for protein precipitation, it also precipitate nucleic acid. And nucleic acid is the culprit of striking in your case.  If you send detail of your protocol for protein isolation, I can suggest the probable changes needed for the same. Also your focusing is adequate so you don’t have to change it.

Regards

@Vivek Ambastha: hey this is an interesting point !!! nothing more Just I isolated the protein and precipitated using methanol: chloroform to remove any nucleic acid/lipid/salts or any interfering substances and after that added the rehydration buffer to it,centrifuged and kept it for rehydration...nothing more than this !!!  hope you are clear what I have explained. Could you suggest me a protocol to remove these interfering substances rather than methanol:chloroform?? will TCA/Acetone works??? and what are you using to make the sample free from contaminants.

Thank you very much

What buffer composition you are using for protein isolation and what is the source of ur protein? 

@vivek: TNE lysis buffer......and did the sample extraction from cell lysate !!!

MDV,

If you are working with mammalian cell lines, then just follow the simple procedure of using rehydration buffer ( 7M urea, 2M thioUrea, 2%Chaps, and DDT) as lysis buffer. The only addition, is you have to sonicate  your sample at 18KHz for 10sec, five pulse followed by centrifugation of your sample at high speed. discard the pellet.  only demerit of this method is , you have to use RCDC reagent for protein quantification.  But prior to lysis , wash your cell pellets to remove media traces remained with your cell culture. 

Vivek: Thank you very much for the suggestions. Since we are using Bradford assay for protein quantification, we didn't do with lysis buffers with excess amounts of urea/DTT and we havent tried the aforementioned method for protein quantification.  Since this is giving a good protein yield without sonication, thought of going with this.