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[Protein-analysis] Re: protein concentration in Ethanol

Dr Engelbert Buxbaum via proteins%40net.bio.net (by engelbert_buxbaum from hotmail.com)
Wed Jan 16 13:41:37 EST 2008


Am 19.12.2007, 16:58 Uhr, schrieb Fery <m.fery51 from gmail.com>:

> I have made a lipid extract from pollen by folch method and dissolve the  
> my lipids in pure Ethanol.  I want to measure the percent of protein  
> contamination in my extract. please let me know what is the best method  
> for measurment of protein in ethanol.

Theoretically there shouldn't be any, since proteins are not supposed to  
be alcohol soluble (and neither in chloroform used during the extraction).  
Short, hydrophobic peptides perhaps.

Their determination would not be easy however, because they could not be  
separated from the lipid by Alcohol/Chloroform or by TCA/DOC, and probably  
not with phenol either. Biuret (and its congeners like Lowry or BCA) may  
not work if the peptides are very short, dye binding neither and aromatic  
amino acids for A280 may not be present.

If I suspected that such peptides were present, I would probably try a 2D  
TLC with Kieselgel G plates (Merck, activated 1 h at 130 deg C and allowed  
to cool protected from humidity) with 1-butanol/acetic acid/water 6+2+2 as  
the first and chloroform/methanol/ammonia 50+40+5 as 2nd dimension mobile  
phase. Detect lipids  by incubating the plates in a closed chromatography  
tank with a few crystals of iodine. Mark the spots with a pencil as the  
colour is not permanent. Then spray with 1 mg/ml ninhydrine in water  
saturated n-butanol and heat for 10 min at 85 deg C. Any purple spots  
except PS and PE would warrant further investigation.


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