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Molecular weight filter and protein concentration

Molecular weight filter and protein concentration - Mass Spectrometry Forum

Molecular weight filter and protein concentration - Discuss and post questions about LC-MS, Q-STAR, MALDI-TOF, and other Mass Spectrometry Questions in the Mass Spec Forum.


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Old 11-10-2010, 10:43 AM
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Smile Molecular weight filter and protein concentration



Hi,

I'm trying to concentrate the fraction from 10 kDa to 30 kDa of my samples in order to analyze them with MALDI-TOF. I tryed AMICON ultra 4 from Millipore, but I'm not satisfyed because when I shot retentates, I found also a lot of protein from 1 kDa to 10 kDa that should flowed to eluate. I can't explain why, and It's a big problem for my analysis.
Someone have suggestions, please ?

Last edited by admin; 11-10-2010 at 11:02 AM.
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Old 11-10-2010, 01:20 PM
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Default Re: Molecular weight filter and protein concentration

Quote:
Originally Posted by Asuriel View Post
Hi,

I'm trying to concentrate the fraction from 10 kDa to 30 kDa of my samples in order to analyze them with MALDI-TOF. I tryed AMICON ultra 4 from Millipore, but I'm not satisfyed because when I shot retentates, I found also a lot of protein from 1 kDa to 10 kDa that should flowed to eluate. I can't explain why, and It's a big problem for my analysis.
Someone have suggestions, please ?
Amicon Ultra 4 is a 3 kDa filter.
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Old 11-10-2010, 05:20 PM
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Smile Re: Molecular weight filter and protein concentration

Quote:
Originally Posted by Asuriel View Post
Hi,

I'm trying to concentrate the fraction from 10 kDa to 30 kDa of my samples in order to analyze them with MALDI-TOF. I tryed AMICON ultra 4 from Millipore, but I'm not satisfyed because when I shot retentates, I found also a lot of protein from 1 kDa to 10 kDa that should flowed to eluate. I can't explain why, and It's a big problem for my analysis.
Someone have suggestions, please ?
I also tried and gave up using Amicon filters for concentration and size separation. I am now using Pierce Slide-a-lyzer (in dialysis cassettes and mini-dialysis units), these work extremely well.
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Old 11-11-2010, 01:07 PM
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Smile Re: Molecular weight filter and protein concentration

Thanks a lot, I'm verty astonished about these filters. Do you know if (in your experience) eletrolyte concentration may modify protein retention capability? If it is I could increase salt concentration to achieve good results. It's very important for me to solve this question.
Bye
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Old 11-11-2010, 04:43 PM
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Smile Re: Molecular weight filter and protein concentration

Quote:
Originally Posted by Asuriel View Post
Thanks a lot, I'm verty astonished about these filters. Do you know if (in your experience) eletrolyte concentration may modify protein retention capability? If it is I could increase salt concentration to achieve good results. It's very important for me to solve this question.
Bye
You don't want to increase salt concentration for two reasons:
1) maldi-tof is sensitive to salts, typically desalt(or dilute) sample before spotting.
2) Dialysis can be used to remove small proteins if the MWCO is 10K. Very efficient with the Slide-A-Lyzer because you use either 100ul (mini) or up to 500ul (cassette) and dialyze against a very large volume like 1L (with an exchange to further pull out the small proteins). That dialysis of small proteins will work despite the ionic difference between buffers.
3) High ion concentration in the spin filters probably doesn't make a difference, since it is all size filtration.

BTW those spin filters are probably getting clogged with proteins (perhaps, glycoproteins, lipids may be in supernatants, depending on sample), thus the retention of the small proteins.
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Old 11-12-2010, 01:49 PM
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Smile Re: Molecular weight filter and protein concentration

Thank you again.
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Old 11-15-2010, 01:21 PM
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Angry Re: Molecular weight filter and protein concentration

Quote:
Originally Posted by danfive View Post
You don't want to increase salt concentration for two reasons:
1) maldi-tof is sensitive to salts, typically desalt(or dilute) sample before spotting.
2) Dialysis can be used to remove small proteins if the MWCO is 10K. Very efficient with the Slide-A-Lyzer because you use either 100ul (mini) or up to 500ul (cassette) and dialyze against a very large volume like 1L (with an exchange to further pull out the small proteins). That dialysis of small proteins will work despite the ionic difference between buffers.
3) High ion concentration in the spin filters probably doesn't make a difference, since it is all size filtration.

BTW those spin filters are probably getting clogged with proteins (perhaps, glycoproteins, lipids may be in supernatants, depending on sample), thus the retention of the small proteins.
I'd like to report some my results. I tryed Amicon 3 kDa filter with different increasing amounts of salts (0-20-40-100 mM of NaCl). After ultrafiltration I desalted with ZipTip. I found that different amounts of proteins were retained or eluted from filter in different salt concentrations.
So that's a real problem If you ultrafiltrate solutions with different salt concentration. Retaining of protein is really modified by sample chemical compositions.
Hope it helps.
Bye.
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Old 02-22-2011, 01:28 PM
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Default Re: Molecular weight filter and protein concentration

I would run the sample on a [Only registered users see links. ]followed by electroelution. It will allow you to separate by size from the gel and select gel portions to be analyzed. You will still want to perform a desalting/cleanup step after electroelution.

If you don't have access to a [Only registered users see links. ]maybe you could run the samples on a gel and perform in-gel digestion of selected bands.

Last edited by proteaSusie; 02-23-2011 at 01:52 PM.
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