In case I could help you,

I use the protocol obtained from the book: Parasite antigens, parasite genes. A laboratory manual for molecular parasitology. Maizels RM, Blaxter ML, Robertson BD and Selkirk ME. In Method 7, SDS-PAGE:

For 2x:

12.5 ml 1M Tris pH 6.8 (final concentration 125 mM)

40 ml 10% SDS (4%)

20 ml Glycerol (20 %)

2 ml 0.2 Bromophenol blue (0.004 %)

Add water to final volume of 100 ml. Store at room Temperature

Where reducing conditions are required, add 5% fresh 2ME

Judging from the image that you posted it's not a problem of which sample buffer you use but the amount of salt in your sample.

In order to get rid of high salt concentrations and concentrate your sample you could try precipitating your proteins in your sample using cold (-20°C) acetone and re-dissolving with your SDS-PAGE sample buffer. Do not use the samples that are already in the SDS-PAGE buffer.

Just google "acetone pricipitation proteins" and you will find the same protocol multiple times. You will have some loss of material but if that is not problem you should get a nicer gel.

Here is a description of the WB issue, I hope you can help you.

https://www.creative-diagnostics.com/resource/pdf/com/Western-blot-troubleshooting-tips.pdf

Hi, Maryam Moazami Goodarzi and all others.

I do agree with Hüseyin Besir and think you may do a few technical actions in order to get rid of it.

1) TCA precipitation is worth to try but may give you some trouble too.

2) Other possibility is pass your sample in dialysis bags or into a desalting column. Both are nice but may be a too high cost (cost X benefit evaluation).

Lastly, as you did not tell us anything about your protocol, sample, electrophoretic parameters, I would recommend one last thing ... run your gel smoothly. No more than 100V or 15mA for each gel (using an equipment like a miniProtean system). Ok?

Because with more resistance (salty sample), current generates more Joule heat that may be giving you, your distorted/strange/bad resolved bands. Ok?

I wish you all the best in your project.

If you need any further assistance, feel free to contact us all again ...

;-)

Hello Dears,

Thank you all for your answers, I performed low voltage and precipitation, they did work.

thanks a lot for your recommendations.