Try a Bradford-type assay, scaled down to microplate format. You can buy reagents pre-made; Pierce makes a good one . It is fast-- you pipet 10 microliters or so from each chromatography fraction into a well with the detection reagent, and wait 5 minutes. If protein concentrations are moderate to high in your peaks, you won't even need to use a plate reader; the fractions with protein will be quite visibly blue against a white background. You would probably want to check the MW of your protein peaks on a gel anyway, but at least you won't need to run lanes with a lot of fractions containing no protein.
Evette S. Radisky, Ph.D. Assistant Professor Mayo Clinic Cancer Center Griffin Cancer Research Building, Rm 310 4500 San Pablo Road Jacksonville, FL 32224 (904) 953-6372 ________________________________ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Sollepura Yogesha Sent: Friday, May 28, 2010 12:04 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein monitoring Dear All, I have expressed 30-40 aa region my protein fused to GST. I subjected it to precision protease cleavage. On the gel I can see the band. When I looked for ProtParam in expasy it shows that my peptide doesn't have Extinction coefficients as " there are no Trp, Tyr or Cys in the region considered, your protein should not be visible by UV spectrophotometry." I need to separate GST from the cleavage mixture. How can I monitor my peptide during FPLC and after that. AA composition is Ala (A) 8, Arg (R) 2, Asn (N) 3, Asp (D) 2, Gln (Q) 2, Glu (E) 2, Gly (G) 4, Ile (I) 1, Leu (L) 4, Lys (K) 4, Phe (F) 2, Pro (P) 2, Ser (S) 5, Thr (T) 5, Val (V) 3. I am looking for some suggestions Thanks in advance Yogi
