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unexpected protein bands

recombinant protein expr

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#1 ocram

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Posted 11 December 2014 - 09:38 AM

I've expressed a protein with a simple 6x His tag in a pET vector.  The DNA sequence is correct and if expressed , the protein should give a band of 25KDa.  I obtain a messy series of bands  on Western blot (anti-His) at around 37KDa and a very faint band at 25KDa.  What does this mean?



#2 bob1

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Posted 11 December 2014 - 09:51 AM

It doesn't mean anything. Normally you need to purify the tagged protein. Multiple banding on a western blot can be the result of a number of things, the more common of which are: primary and/or secondary antibody concentrations wrong, secondary incubation too long, and not enough washing. Then you can get into things like not denaturing the proteins enough, too much denaturation (causing aggregation), too much protein loaded... etc.



#3 labtastic

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Posted 29 December 2014 - 10:06 AM

As bob suggested, purify (or at least enrich) the protein with some nickle or cobalt resin.

 

Run a gel on the eluted protein and stain with coomassie. When you run your protein, run it at least 4 different ways, (i) not boiled without reducing agent (e.g. 4% mercaptoethanol), (ii) not boiled with reducing agent, (iii) boiled without reducing agent and (iv) boiled with reducing agent. Load ~5-10ug of protein in each lane. Use TCA or acetone precipitation if your eluted protein saple is too dilute to get that much protein in your gel.






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