7 replies to this topic

[Anna1984]

Hi,

I have the problem that I have a protein sample in a solution containing 200mM NaCl.
I am afraid this high salt concentration interfereswith the reducing agents since I have problems getting clear results with SDS Page and WB.

For further samples (which come from cell fractionations) I willu se a different lysis buffer, but until then I would like to save some of my sample that I have so far. Also to try out if this is the only problem in my WBs.

Does anyone know a quick way of changing the buffer of my protein sample, maybe precipitation? I unfortunately have not a lot of resources at hand... So no columns or dialysis.

Thanks a lot!

[K.B.]

Acetone or ethanol precipitation, perhaps?

Or Three Phase Partitioning - if you have t-butanol and ammonium sulphate?

[darkrxn]

if you don't have any neighbors that will give you one of their, "zip-tips," (which are like pipette tips so you get 96) then maybe somebody will loan you a 5kDa MWCO spin down concentrator? I use them in a pinch, its clearly not as smart as using a column or desalting pipette tip, but its fast and easy and great whole cell lysate yields.

[darkrxn]

I use 150mM NaCl and 0.1% SDS for cell lysis and the salt does not affect my WB, though.

Edited by darkrxn, 30 July 2009 - 03:04 PM.

[T C]

Hey,

Just use a desalting column.  PD-10 from GE works very well.  

http://www1.gelifesc...;ModuleId=42278

Best,
TC

Anna1984, on Jul 30 2009, 10:25 PM, said:

Hi,

I have the problem that I have a protein sample in a solution containing 200mM NaCl.
I am afraid this high salt concentration interfereswith the reducing agents since I have problems getting clear results with SDS Page and WB.

For further samples (which come from cell fractionations) I willu se a different lysis buffer, but until then I would like to save some of my sample that I have so far. Also to try out if this is the only problem in my WBs.

Does anyone know a quick way of changing the buffer of my protein sample, maybe precipitation? I unfortunately have not a lot of resources at hand... So no columns or dialysis.

Thanks a lot!

[chandrajit]

hey my problem is somewhat different......
i have peptides and some salts in it........i want to go ahead for maldi....
the problem is......when i do desalting using revesre phase spin columns by pierece....the spectra i get after the analysis  is not as clean as it should be like the one's we commonly see in research papers using the same...and not all the peptides showup, there is loss of peptides as well (may be the column didn't bind to the peptides and we lost them)
can someone help me in atleast getting a clear spectra......il know there wiill be som peptide losses...that can't be solved

[K.B.]

chandrajit,
I prefer gravity flow SPE columns so I can control what's going on.
In your case I would run "blank" sample (ie. water, buffer or no sample at all) before your sample, to remove any impurities that may be already present on the column.
What buffers/solutions do you use for column equilibration, wash and elution?

[colic101]

Hi,thanks for posting. I was about to make the same thread as yours. Nice thing i've bumped into your thread. Anyways im still looking for further suggestions. Could anyone here help me out? Thanks!


Regards,
colic101
Placement financier