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Protein bands not migrating

SDS-PAGE Silver stain

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3 replies to this topic

#1 Hakusan1

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Posted 21 September 2012 - 08:41 AM

Greetings,

I've been doing some silver staining recently and some of the bands in my gels have just stopped migrating. They cross the border between the stacking and resolving gel just fine but then seem to stop migrating. I've cast new gels and changed sample buffers. The molecular weight markers are migrating just fine, any suggestions as to what's causing this. This is a new phenomenon so any help resolving it would be a big help.

#2 ascacioc

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Posted 21 September 2012 - 11:18 AM

I have this when I have protein aggregates in my samples; add more SDS or/and DTT/mercaptoethanol in the sample buffer. Some sample need a bit more of those.

#3 mdfenko

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Posted 25 September 2012 - 08:32 AM

you may be boiling the sample too long. if you want to boil for more than 3-5 minutes then you could incubate at 60-70C for 10-20 minutes.

this is assuming that you are performing sds-page and using sds sample buffer.

if you are performing native page then you should not be warming you samples at all. just add sample dye (bromphenol blue) and sucrose or glycerol (to increase density) and load. in this case your sample may aggregate due to age or handling.
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#4 cbf88

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Posted 29 September 2012 - 01:17 AM

adding antioxidant to the gel chamber can help in addition to everything else mentioned




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