Hi Fereshteh,

First you have to discriminate whether this happens already during the run, or only during staing (diffusion of the proteins). If this happens during the run, it my be due to a) an old gel matrix / too old reagents or b) too high salt content in your samples or c) too old/not optimized gel loading buffer. If it happens during staing, it may be due to welling of the gel matrix and your need to add more methanol/ethanol, in order to keep the gel in its form.

Try some quick stain without the need to destain your gels (e.g. Roti-Blue quick). It might help discussion if you could attach some picture of a typical gel ...

Stefanie

Pre-cast or home made gel? If you casted gel yourself then you need to make sure all the ingredients are added sequentially in the right way. Make sure you use the right buffer pH. If you used pre-cast gel then make sure the running buffer has right pH. Also, make sure you do spin down the junk pellet if fail to do so then the separation will be messed up.

Probably you overloaded the gel wells

Hi Fereshteh,

First you have to discriminate whether this happens already during the run, or only during staing (diffusion of the proteins). If this happens during the run, it my be due to a) an old gel matrix / too old reagents or b) too high salt content in your samples or c) too old/not optimized gel loading buffer. If it happens during staing, it may be due to welling of the gel matrix and your need to add more methanol/ethanol, in order to keep the gel in its form.

Try some quick stain without the need to destain your gels (e.g. Roti-Blue quick). It might help discussion if you could attach some picture of a typical gel ...

Stefanie

Hi

I think sample diffusion occurs, this may be due to heat produces upon voltage drop or flucctuation.

Changes in buffer pH may be the cause of heat production.

If u cast the gel with high porosity for determining high molecular weight protein the low molecules may diffuse to near by wells due to voltage fluctuation or drop.

overloading of sample leads to clumping and we cant see the clarity of the band but it never diffuses the well

chk the buffer pH

voltage (CC mode till dye reaches separating gel; CV mode till reaches the bottom)

If you need the protocol send me ur mail .

Thank u

Hi Noorjahan

thank you for your guide.

my mail: [email protected]

but I can't understand how can buffer ph affect diffusion my sample and then cause my sample combine with each other?

hi Stephanie

thank you for your guide.

How can process of staining cause diffusion my protein?

How can I understand which reasons happen for this problem

I attach my photo, at last tow well, I run samples as two well beside them is free. yo can see how my band of sample is wided

Hi,

Now, I`m sure that you overloaded your gel, particularly first three lines on the left. The bands from the samples on the right wided because between them nothing was loaded. Next time, if you want to separate your two samples with the free well, just load a 1x loading buffer (the same added to your samples) to the "free well".

Hi, sure overloaded and voltage fluctuation too occurs. I send you a protocol which you can follow.

Gud luk

Hi fereshteh

there are three thing u take care:

1) load less amount of protein

2) run gel at constant voltage and current maximum.

3) heating effect is possible cause and to avoid it always load just dye at the empty end wells of ur gel and empty well in between ur sample.

Take care of three point and ur gel show very good band

gud luck

regards

ravindra

Hey Fereshteh,

The process of stainig itself does not cause this phenomenon. However, if the staining solution is made from reagents that are too old you may get this swelling of the gel / bands. Particularly the age of methanol is VITAL! So, check your methanol and only use bottles of 1 year age in maximum.

Please also check your protein extract / loading buffer for salt content. I once got this phenomenon in a customer's gel, who used extracts from marine organisms and had very high salt contents in his samples. You may have to desalt a bit prior to your gel.

Definitely also use MUCH less extract as loading samples. Prepare one gel with a dilution series of 1:2 each (1:2, 1:4, 1:8, 1:16, etc.) in order to get some idea on how to dilute best.

Good luck,

Stefanie

thank you for all your guide, finally I could get good gel!