SDS PAGE Double Bands for >6 months - (Mar/23/2008 )

Greetings,

Our lab has has successfully used SDS PAGE gels for MHC determination for the past decade. We typically use a 5% separating with a 3.5% stacking and run for 12-18 hours at 150 V (constant). We fix it in acetic acid/ethanol solution, expose it to gluteraldahyde, followed by silver stain and developer. Over the past six months, most of our bands (200 kD) are showing up with an almost 3D effect and appear as two bands or a double band. We have changed out all of our chemicals, purchased a new apparatus, and replaced all of the old accessories (combs, plates, etc.). We have also ran old tissue that we know is good, and that showed the same phenomena. We have also tested temperature regulation and that doesn't seem to be the issue. To our knowledge, nothing in our protocol has deviated from what had been implemented over the past decade. The challenge here is that they are inconsistent. Our sample tissue is solubilized in sample buffer and is then stored at -20. When the sample is ready to be run, the sample is boiled and a small amount is aliquoted while the remaining sample is stored at -20. There are inconsistencies within a sample. For example, an aliquot doesn't always show the double band. Further, an entire gel with different samples generally contains some lanes that double band and others that do not.

Is there any chance that something that we are doing after the proteins have migrated could be causing this double band effect?


We feel as if we have tried everything. Thank you so much for your time.

Any thoughts?

Hey, what about the protein marker? Is that running properly? Have you tried another protein marker?

Maybe something about the water you are using in the lab (the pH)?

Sorry, I cannot think of anything else, either. You seem to have tried almost everything.

Let`s hope that it works again!

Would you be so kind and give me your gel recipe ? We are running 6% big SDS gels to look at two proteins at ~200-250 kDa. However, during running all the marker bands swop position or they disappear although we put more of the marker. I cannot talk for the samples because they seem to not be transferred properly for Western. However, without the marker nothing helps.

how old are your stocks (specifically, your 2-me or dtt and other buffer components)?

have your glass plates warped slightly? this would cause a loose fit for the comb which will allow gel to polymerize higher than the bottom of the well (which could hold back some of the sample).

just a couple of things to check.

Hey, what about the protein marker? Is that running properly? Have you tried another protein marker?

Maybe something about the water you are using in the lab (the pH)?

Sorry, I cannot think of anything else, either. You seem to have tried almost everything.

Let`s hope that it works again!

Would you be so kind and give me your gel recipe ? We are running 6% big SDS gels to look at two proteins at ~200-250 kDa. However, during running all the marker bands swop position or they disappear although we put more of the marker. I cannot talk for the samples because they seem to not be transferred properly for Western. However, without the marker nothing helps.

Check your PM