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Alcian Blue and Silver Staining for CPS and LPS


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#1 Suziebee

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Posted 21 August 2012 - 04:36 AM

Hi guys,

I'm wondering could someone help me with interpreting my SDS-PAGE gel.Posted Image

I am looking for the presence of a Capsular Ploysaccharide on my bacterium, so I ran a 12% SDS-PAGE with freshly harvested cells, washed twice with PBS, and resuspended in Saline and heated at 50°C for 1hour (CPS is thermostabile) and centrifuged. The supernatant was removed and boiled with sample buffer for 5mins and loaded onto the 12% sds-gel.

I stained the gel with alcian blue and have discrete bands around 70-100kDa. There is no mAb fro the CPS of this bacteria so I can't do Western Blots.

Does anyone know how I can confirm this is a CPS or if not what else it may be?Posted Image

Thanks for reading this and any advice is much appreciated!!

SusanPosted Image

#2 bob1

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Posted 21 August 2012 - 12:56 PM

If you have a stained band you can do some mass spectrometry on it to confirm. Unfortunately there isn't really any other method of confirmation, other than using an antibody or doing edman degradation (very labour intensive and expensive).

#3 Suziebee

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Posted 22 August 2012 - 08:16 AM

Hi Bob,

Thanks for your reply but unfortunately we don't have a mass spec at our ease.

Do you know from your experience if the epitopes for CPS antibodies are pretty conserved across species?

Thanks,
Susan

#4 bob1

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Posted 22 August 2012 - 12:38 PM

No idea sorry.




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