hi, folks
I have horizontal tails with the protein spots on my 2D tricine PAGE, any one have a good solution?
thx

I hope you might be running perfect 2-D gels with single spots and wish you to share your experience with us.

Here attached a pdf describing probable reasons and solutions to horizontal & vertical streaking in 2-D gels.
Thank you.

I do run decent native-2D gel, willing to share with anybody who interested.

btw, seems the PDF file has a password control, any chance that you have the passcode

I have attached one more .
Prev. one I will send you if you want that was from Biorad trouble shooting guide for 2D.

But would really like to know the problem practically you had and how you resolved it.
Thank you

Iam facing a problem in downloading from here once attached never faced before will send in your mail.

Horizontal Streaking or Incompletely Focused Spots:
1.Make sure that the sample is completely and stably solubilized- Repeated precipitation-resolubilization cycles produce or increase horizontal streaking.Increase the concentration of the solubilizing components in the rehydration solution.
2.Interfering substrates- Nonprotein impurities in the sample can interfere with IEF, causing horizontal streaking in the final 2D result, particularly toward the acidic side of the gel.-Modify sample preparation to limit these contaminants.
3.Ionic impurities in sample- Reduce salt concentration to below 10 mm by dilution or desalt the sample by dialysis. Precipitation with TCA and acetone and subsequent resuspension is another effective desalting technique that removes lipids, nucleotides, and other small molecules. Specific and nonspecific losses of proteins can occur with dialysis, gel chromatography, and precipitation/resuspension of samples. If the sample cannot be modified, reduce the effect of ionic impurities by modifying the IEF protocol. Limit the voltage to 100–150 V for 2 hours, then resume a normal voltage step program. This pre-step allows the ions in the sample to move to the ends of the IPG strip.
4.Ionic detergent in sample- If the ionic detergent SDS is used in sample preparation, the final concentration must not exceed 0.25% after dilution into the rehydration solution. Additionally, the concentration of the nonionic detergent present must be at least 8 times higher than the concentration of any ionic detergent to ensure complete removal of SDS from the proteins.
5.High sample load - Load less sample. Micropreparative separations require clean sample. Modify sample preparation to limit concentrations. Program a low initial voltage gradually. Extend focusing time.
6.Underfocusing. Focusing time was not long enough to achieve steady-state focusing-Prolong focusing time.
7.Overfocusing. Extended focusing times (>100,000 Vh) may result in electro-endosmotic water and protein movement, which can produce horizontal smearing-Reduce focusing time.


Finally pasted here.

hi, Mchinmoyee, yes, actually I figured it out, I was running Blue Native-2D gel of membrane proteins, to get a good visualization on the BN gel, I need large amount of proteins, which is not very compatible with the 2nd-D SDS-PAGE, and made the horizontal stripes of high molecular weigh proteins.

thanks, but still, I have problem opening the PDF file (seems to password protected).

Thank you for sharing.
So the problem was due to high protein loading?

yes, it was too much over loading of proteins, which was good for Blue native detection (with CBB G250) but not suitable for the 2D running and detection