Re: [ccp4bb] Troubleshooting protein purification cation IEX
Hi, Many things can lead to your observation. Please outline all steps of your purification procedure as it is not clear what is done before and after the Ion Exchange steps. I am not sure if IEF in your emails refers to Isoelectric focusing, as the acronym is usually used?? Couple of suggestions: 1) Instead of contamination, you might just be seeing multiple bands due to 'aggregation' of your protein! Make sure you boil the sample prior to loading on gel and also that your loading dye contains SDS, bME/DTT. 2) We used to do entire purifications with inclusion body preps under denaturing conditions to prevent unwanted aggregation of partially folded or misfolded species. Not sure if denaturant is present all along. 3) If the problem is of contamination, try making the gradient shallow for the 35 –80% gradient step in Ion Exchange (increase to say, 20 cv or more). 4) If the problem is of contamination, try to add more steps to purification -- e.g., affinity step (if possible), anion exchange as well etc. 5) If IEF (in the sense I mean it) is what you did and it shows only 1-2 bands, the problem is likely (#1) outlined above. 6) If all else fails, cut out one or two of the bands from your gel and run a mass spec. An expensive way to find out that it is aggregation. Nevertheless Hope that helps. Raji On Sep 23, 2008, at 3:51 AM, Meg wrote: Dear All, This is with reference to the purification of our recombinant protein sample expressed in E.coli as inclusion bodies. After Solubilization refolding we perform the cation exchange chromatography of our protein sample using SP sepharose fast flow resin. Attached herewith find the SDS PAGE and IEF results of the collected fractions. In addition to our protein of interest we are also getting high molecular weigh contaminants, which we cannot get rid of in IEX. Can anyone please guide me on a technique to get rid of these bands as even after gel filtration of samples few high mol wt contaminant bands are not separated from main proteins and sample gets diluted too. In cation IEX procedure is Column Sp Sepharose Fast flow packed in fineline 35 column packed bed volume 100 ml System AKTA FPLC Equilibration 10 C.V. protein loading 3-4 C.V. [approx 1mg/ml protein conc], washing to remove unbound materials 2 C.V. step elution 0-35% gradient – 1 C.V., 35 –80% gradient – 10 C.V. and 60 –100% gradient 1 C.V. Protein elutes at 40-50% gradient. Protein details: Our protein is stable at acidic pH and has a pI of 5.8 –6.3 and buffer is Na Acetate buffer pH 4.5 and elution buffer is starting buffer containing 0.4 M NaCl. We get only one peak on AKTA but on running SDS page we get so many bands even IEF shows 1-2 bands at the most. How can we modify the method or what can be done to get rid of extra high mol wt bands. Any help will be deeply appreciated. SDS PAGE.JPGIEF.doc
Re: [ccp4bb] Troubleshooting protein purification cation IEX
Hello Meg, Since your protein is quite acidic, your next step could be e.g. anion exchange - provided that you are able to get the protein into a suitable buffer w/o losing it (since you will pass through the pI). If you can, the simplest way to do so is to add TRIS to the pH4.5 buffer until you get the desired pH (7-8) and dilute the sample a bit with pure water. An alternative, or a follow-up step could be hydrophobic column - these are very useful for removing aggregates, partially folded stuff and similar entities. Again, nothing is completely safe - HIC may cause problems since you are starting at fairly high salt (1-2 M NaCl or KCl typically). But at least you won't have to pass the pI. Cheers, Artem Dear All, This is with reference to the purification of our recombinant protein sample expressed in E.coli as inclusion bodies. After Solubilization refolding we perform the cation exchange chromatography of our protein sample using SP sepharose fast flow resin. Attached herewith find the SDS PAGE and IEF results of the collected fractions. In addition to our protein of interest we are also getting high molecular weigh contaminants, which we cannot get rid of in IEX. Can anyone please guide me on a technique to get rid of these bands as even after gel filtration of samples few high mol wt contaminant bands are not separated from main proteins and sample gets diluted too. In cation IEX procedure is Column Sp Sepharose Fast flow packed in fineline 35 column packed bed volume 100 ml System AKTA FPLC Equilibration 10 C.V. protein loading 3-4 C.V. [approx 1mg/ml protein conc], washing to remove unbound materials 2 C.V. step elution 0-35% gradient 1 C.V., 35 80% gradient 10 C.V. and 60 100% gradient 1 C.V. Protein elutes at 40-50% gradient. Protein details: Our protein is stable at acidic pH and has a pI of 5.8 6.3 and buffer is Na Acetate buffer pH 4.5 and elution buffer is starting buffer containing 0.4 M NaCl. We get only one peak on AKTA but on running SDS page we get so many bands even IEF shows 1-2 bands at the most. How can we modify the method or what can be done to get rid of extra high mol wt bands. Any help will be deeply appreciated.
Re: [ccp4bb] Troubleshooting protein purification cation IEX
Meg, I might add that if you have boiled your samples, you might disregard this: 1) Instead of contamination, you might just be seeing multiple bands due to 'aggregation' of your protein! Make sure you boil the sample prior to loading on gel and also that your loading dye contains SDS, bME/DTT. But multiple bands might be due to aggregation of your protein BECAUSE of boiling the samples. This is common with some membrane and somewhat hydrophobic proteins. Not boiling or not heating your samples may reduce it. By the way, how do you know that you have refolded your protein specifically? Do you have an activity assay? Refolding can yield aggregates like protein micelles: soluble in the sense that they don't precipitate, but still aggregates that can include other proteins. Can you purify your target protein while denatured? That may solve part of your problem. Good luck, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: [EMAIL PROTECTED] On Sep 23, 2008, at 7:54 AM, Raji Edayathumangalam wrote: Hi, Many things can lead to your observation. Please outline all steps of your purification procedure as it is not clear what is done before and after the Ion Exchange steps. I am not sure if IEF in your emails refers to Isoelectric focusing, as the acronym is usually used?? Couple of suggestions: 1) Instead of contamination, you might just be seeing multiple bands due to 'aggregation' of your protein! Make sure you boil the sample prior to loading on gel and also that your loading dye contains SDS, bME/DTT. 2) We used to do entire purifications with inclusion body preps under denaturing conditions to prevent unwanted aggregation of partially folded or misfolded species. Not sure if denaturant is present all along. 3) If the problem is of contamination, try making the gradient shallow for the 35 –80% gradient step in Ion Exchange (increase to say, 20 cv or more). 4) If the problem is of contamination, try to add more steps to purification -- e.g., affinity step (if possible), anion exchange as well etc. 5) If IEF (in the sense I mean it) is what you did and it shows only 1-2 bands, the problem is likely (#1) outlined above. 6) If all else fails, cut out one or two of the bands from your gel and run a mass spec. An expensive way to find out that it is aggregation. Nevertheless Hope that helps. Raji On Sep 23, 2008, at 3:51 AM, Meg wrote: Dear All, This is with reference to the purification of our recombinant protein sample expressed in E.coli as inclusion bodies. After Solubilization refolding we perform the cation exchange chromatography of our protein sample using SP sepharose fast flow resin. Attached herewith find the SDS PAGE and IEF results of the collected fractions. In addition to our protein of interest we are also getting high molecular weigh contaminants, which we cannot get rid of in IEX. Can anyone please guide me on a technique to get rid of these bands as even after gel filtration of samples few high mol wt contaminant bands are not separated from main proteins and sample gets diluted too. In cation IEX procedure is Column Sp Sepharose Fast flow packed in fineline 35 column packed bed volume 100 ml System AKTA FPLC Equilibration 10 C.V. protein loading 3-4 C.V. [approx 1mg/ml protein conc], washing to remove unbound materials 2 C.V. step elution 0-35% gradient – 1 C.V., 35 –80% gradient – 10 C.V. and 60 –100% gradient 1 C.V. Protein elutes at 40-50% gradient. Protein details: Our protein is stable at acidic pH and has a pI of 5.8 –6.3 and buffer is Na Acetate buffer pH 4.5 and elution buffer is starting buffer containing 0.4 M NaCl. We get only one peak on AKTA but on running SDS page we get so many bands even IEF shows 1-2 bands at the most. How can we modify the method or what can be done to get rid of extra high mol wt bands. Any help will be deeply appreciated. SDS PAGE.JPGIEF.doc
Re: [ccp4bb] Troubleshooting protein purification cation IEX
Another suggestion that comes to mind is crosslinking via Cys, are you purifying under reducing conditions ? Or since you have inclusion bodies they're in general not correctly folded hence expose hydrophobic regions and can interact with other proteins, to avoid unspecific interaction add 20% glycerol to your lysis buffer. Jürgen On 23 Sep 2008, at 00:51, Meg wrote: - Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
Re: [ccp4bb] Troubleshooting protein purification cation IEX
Another option is to purify in denatured condition, then refold. --Chun -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Meg Sent: Tuesday, September 23, 2008 12:51 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Troubleshooting protein purification cation IEX Dear All, This is with reference to the purification of our recombinant protein sample expressed in E.coli as inclusion bodies. After Solubilization refolding we perform the cation exchange chromatography of our protein sample using SP sepharose fast flow resin. Attached herewith find the SDS PAGE and IEF results of the collected fractions. In addition to our protein of interest we are also getting high molecular weigh contaminants, which we cannot get rid of in IEX. Can anyone please guide me on a technique to get rid of these bands as even after gel filtration of samples few high mol wt contaminant bands are not separated from main proteins and sample gets diluted too. In cation IEX procedure is Column Sp Sepharose Fast flow packed in fineline 35 column packed bed volume 100 ml System AKTA FPLC Equilibration 10 C.V. protein loading 3-4 C.V. [approx 1mg/ml protein conc], washing to remove unbound materials 2 C.V. step elution 0-35% gradient - 1 C.V., 35 -80% gradient - 10 C.V. and 60 -100% gradient 1 C.V. Protein elutes at 40-50% gradient. Protein details: Our protein is stable at acidic pH and has a pI of 5.8 -6.3 and buffer is Na Acetate buffer pH 4.5 and elution buffer is starting buffer containing 0.4 M NaCl. We get only one peak on AKTA but on running SDS page we get so many bands even IEF shows 1-2 bands at the most. How can we modify the method or what can be done to get rid of extra high mol wt bands. Any help will be deeply appreciated.
