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No protein lane have bands on Coomassie and western blot?!


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4 replies to this topic

#1 Papermate

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Posted 17 October 2010 - 01:50 PM

Hi everyone,

I'm trying to detect 6 kDa protein from my bacteria cell lysate and supernatant (background: I'm testing for successful secretion and overexpression of my protein into the supernatant). I've been running 12% SDS gels (reducing conditions) and so far have been unsuccessful after making fresh buffers, new samples, loading less sample etc.

Problem:

In my empty lanes (sample buffer + water), I still get bands (seen in Coomassie staining and Western blot) when I know there are no protein in those empty lanes. I have absolutely no idea why. These odd bands are masking my protein positive control (35 ng and 50 ng) and possibily my protein of interest... please help!

Any thoughts/suggestions would be greatly appreciated! Thanks!

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#2 bob1

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Posted 17 October 2010 - 04:39 PM

Dust in your sample buffer - do you wear gloves during preparation? Dust is about 60-70% keratin from keratinocytes shed from your skin.

#3 GNANA

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Posted 18 October 2010 - 03:46 AM

The bands in the empty lane could be the overflow from ur lysate loaded lanes. you are saying 6 kd but i think the marker u have shown stacked at 10 KD increase ur PAGE conc. may be 15 percent might help. good luck..

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#4 mdfenko

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Posted 18 October 2010 - 06:59 AM

the buffer front seems to be staining.

for a 6kDa protein i would use a tris-tricine buffer system with a 10% gel for separation.
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#5 Papermate

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Posted 23 October 2010 - 01:40 PM

Hi all,

Thank you for your suggestions.

I've tried increasing my SDS gel concentration to 15% (and ran the dye front off) and still get the same results. Since I know the contents of the dye front is not interfering with my proteins, it is possible that my proteins are still not being fully resolved... but I have one question, since I'm using purifed commercially bought protein samples for my positive controls (at 15ng, 30ng, 60ng), shouldn't they be easily 'resolved' though? My positives have showed up on the western blots before when I ran 12% gels (Tris-Glycine), but not anymore!

People in my lab have ran proteins from 5 kda - 10 kda before using the same protocol + conditions and have no trouble separating and detecting proteins... so I'm totally lost right now.

Please throw out any suggestions/tips you have! Thanks!




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