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Help Me With Polyclonal Production


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8 replies to this topic

#1 ilovebacteria

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Posted 29 November 2010 - 12:16 PM

Can someone please recommend best practice or a recommended method to help with the clotting of rabbit serum. We are getting occasional extra bands at 80 kda and it is unexplained. We think that maybe it is because we are clotting our rabbit serum wrong.

What we did:
We purified rabbit serum and ran an SDS page and notices that some of purifications has extra third band around 80 kda (not from the light and heavy chains). Can someone please help to explain why this is happening?
Could it be because of the way we are bleeding the rabbits or the way we are collecting the serum? What is the best way to do this? Has anyone else had an extra band in their serum?

Thanks!

#2 Chelo

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Posted 29 November 2010 - 01:33 PM

It looks as if you were getting the whole antibody as well. Are you sure the reducing conditions of your running buffer are ok?

#3 ilovebacteria

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Posted 29 November 2010 - 01:44 PM

Conditions of the running buffer are fine. We are positive it's not the whole antibody.

#4 protolder

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Posted 29 November 2010 - 10:37 PM

Hola, I think that if the serum hasnīt be purified, the band could correspond to serum albumin, that is very abundant
in all the sera, and has a MW in the range that you say. Good luck

#5 ilovebacteria

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Posted 30 November 2010 - 11:05 AM

The serum has been purified...

#6 ilovebacteria

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Posted 30 November 2010 - 11:12 AM

It is not serum albumin.

#7 sgt4boston

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Posted 01 December 2010 - 03:25 AM

Please explain how you are 'purify' your antibody.

#8 Chelo

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Posted 10 December 2010 - 12:16 AM

Please explain how you are 'purify' your antibody.

Another possibility is IgM, it has a molecular weight around that value.

#9 sgt4boston

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Posted 10 December 2010 - 03:20 AM

IgM is much larger than G...if you did ammonium sulfate cut and fractionated by size close bands would be albumin and transferrin. If you used affinity chromatography then you should have a pure product. You can test the band with labelled antibody for possible contaminants or run an Ouchterlony double diffusion.




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