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Problem: peptide (MW 2.3KDa) can't dialyze out

peptide dialysis

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5 replies to this topic

#1 huangh84

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Posted 24 February 2012 - 07:18 AM

Hi, all,

I need help figuring out a simple lab dialysis problem involving a peptide I am using.

I am trying to test whether I can use dialysis to remove the peptide (MW 2300) dissolved in PBS pH7.4 (home made). Thought I would try a postivie control first by using the 50,000 MWCO RC dialysis tubing from spectrumlabs and is dialyzing against PBS pH7.4 (home made) at 1:4000 volume ratio. The dialysis is under 4C overnight.

I find almost no loss of the peptide the next day...

Any help is totally appreciated as I am a total rookie on peptide and related subjects.

Thanks in advance!

H.

P.S. I am trying to perform scaled up production involving this peptide so I figure dialysis is probably the best way to remove the excess peptide for big volume production.

#2 mdfenko

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Posted 24 February 2012 - 12:40 PM

are you sure that the peptide is in monomer form?

is the dialysis stirred?

have you checked the dialysis buffer for the peptide?

have you considered ultrafiltration instead of dialysis?
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#3 huangh84

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Posted 25 February 2012 - 08:13 AM

are you sure that the peptide is in monomer form?

is the dialysis stirred?

have you checked the dialysis buffer for the peptide?

have you considered ultrafiltration instead of dialysis?


Hi, thanks for your reply.

I checked with Fast Protein Liquid Chromatography in our lab on the peptide in PBS, and the elution time suggest that it is a small molecule. Whether it is in monomer form I haven't checked.

The dialysis is stirred.

I haven't checked the dialysis buffer for this peptide since this is a custom made one (but how do you check the dialysis buffer of a peptide? Lit search or there is a standard website?). I assumed using the same buffer it is dissolved in for its dialysis made sense.

Ultrafiltration works through centrifugation with 10K MWCO. But as I said, I would like to scale up the process and ultrafiltration may not be feasible.

Thanks,

H.

#4 mdfenko

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Posted 27 February 2012 - 08:36 AM

you should check the dialysis buffer for your peptide the same way you check the sample. dialysis is an equilibrium driven process so some will always be left behind.

you can ultrafilter with stirred cells and can increase process volume with a reservoir (they used to have a bigger one available but don't show it now, it may be available through a used lab equipment vendor).
talent does what it can
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#5 huangh84

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Posted 28 February 2012 - 08:11 AM

Thanks for your reply.

I didn't find any peptide in the dialysis buffer (or they are too low to be detected).

I wonder if TFF will work?

#6 mdfenko

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Posted 29 February 2012 - 01:08 PM

tff could work but so should dialysis and tff is just a fancy dialysis method.
talent does what it can
genius does what it must
i do what i get paid to do





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