During sample preparation for SDS-PAGE, some of my samples are precipitating metimes. They give smear like band on gel, what could be the reason?
Heating at 95 for 5min and then cooling on ice for 3-5 min, buffers are same as generally used, pH is also maintained.
Hi,
Do you have KCl in your sample? if yes, it forms KDS and forms precipitates
Thanks Laxmi for ur reply, yes KCl is there, so, what is the solution to avoid this.
what is the buffer composition? what kind of protein are you working with?? check if you can replace KCl with NaCl. protein degradation or precipitation is your problem..explain ..
Which extraction/purification method you are using for your proteins?
I wonder if your protein is purified properly, sometimes presence of contaminating agents may lead to denaturation of proteins. Or may be some salts are still present in the protein mix that is leading to precipitation in your samples.
Hi Rakesh,
If you are extracting membrane protein, then heating at 95 deg will give more aggregates, and it will not give proper banding in gel. Also, if your SDS concentration goes high, you will get this smear in the gel. Check your salt as well as SDS concentration.
All the best
Regards,
Rajkumar
Agreed to everyone above regarding salt and SDS. One possibility is the technique u use to grind your cells. If it is by sonication, u might consider reducing the sonication time. If its is mechanical crushing by beads in presence of lysis buffer, excessive agitation might break the proteins. Also, if they are tumour samples than its is difficult to get rid of cellular lipids which can create this problem.
Try using sodium deoxycholate in the lysis buffer, which can remove the protiens from the cell membranes and than you can precipitate only the proteins from the aqueous solution and remove other contaminating cell debris.
While adjusting the pH of the buffer, take care not to add excessive acid and base to bring the target pH. Caliberate the pH meter before you completely rely on it.
Try loading lesser protein amount and see if you can get rid of smearing effect.
Most important thing, add protease inhibitor cocktail along with EDTA and prepare your protein on ice with cooling centrifuge.
Membrane proteins form aggregates when boiled at 95C, so what we do is a mild denaturation by incubating samples at 37C for 30-60 minutes. This work well for membrane proteins. Also, you can add 1x Laemmli sample buffer directly to the cells if you are working with lysates.
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