Re: [ccp4bb] protein monitoring
4. protein monitoring Dear Yogesha, You can use the absorbance at 215 or 220 nm to follow your peptide during purification. Compounds like DTT and EDTA can increase the noise at those wavelengths, go to 220 nm if you have things like that in your purification buffers. Quantification of the peptide will have to be performed by Bradford or other colorimetric method, and I guess you can use that quantification to find out the extinction coefficient of your peptide at 215-220 nm. Luck, Carlos
Re: [ccp4bb] protein monitoring
Any protein will absorb at 230, and most UV detectors these days can reach that, if you want to follow that with UV. Together with SDS_PAGE of course, this should be good enough to go ahead. A. On May 28, 2010, at 18:03, Sollepura Yogesha wrote: 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
[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
Re: [ccp4bb] protein monitoring
Hi Yogi, You can see your peptide on a gel so why can't you monitor it by SDS-PAGE? A little time consuming, yes, but then you have the extra benefit of also seeing if there are contaminating proteins in your sample. good luck, Eric __ Eric Larson, PhD MSGPP Consortium Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 On Fri, 28 May 2010, Sollepura Yogesha wrote: 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, I le (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
Re: [ccp4bb] protein monitoring
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
