Hi Need your help! I am performing a 2d gel electrophoresis of chronic myeloid leukemia, but the problem is horizontal streaking’s are appearing in the gels although I tried all sorts of trouble shooting, please help regarding this issue?
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Daniel
The most common cause for horizontal streaking in 2DE gels is salt that leads to insufficient Isoelectric Focussing. It can also be caused by excess protein loaded and less availability of ampholytes etc. You can add a TCA/ Acetone precipitation step in your protocol. That should resolve the issue. I also recommend using CHAPS or CHAPSO along with TBP and adding extra ampholytes to your gel. 2DE is a tough technique and takes years to master.
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Daniel Suarez
Argerine has covered it well, but I would also like to add another point that is about the sample buffer you are using for the loading of proteins on IPG Strips. Majority of times, the solubilization buffer itself contains NaCl and If these buffers contain salt than that is again added salt for the sample analysis. Also what is the source of your protein? A liquid sample always have salt from source e.g. Plasma, Urine, CSF
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That’s correct Biographix. I specialize in Blood Plasma Proteomics and optimization of 2DE was tough due to inherent salt present in plasma. In our case though an ice cold protein precipitation with TCA/Acetone followed with a combination of detergents CHAPS, CHAPSO, NP40, substitution of DTT or DTE with TBP and additional ampholyte concentration helped. Sample solubilization can be challenging in case the temperature control has not been maintained well due to protein denaturation.
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Daniel
Since you are working with cells, you can also check for the Nucleic Acid Contamination: Nucleic acids increase viscosity and cause streaking. Treat with DNase/RNase. In addition to that, check for the Hydration status of your strip Poor Strip Rehydration Allow sufficient rehydration time (overnight at room temperature).
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