Re: [ccp4bb] [OT] which column to use in SLS/MALS instruments
Regarding to amount of material loaded, I think it may also depend on the buffer. When I did light scattering experiment with membrane protein at angle of 90 degree, I got much better base line with buffer containing DDM than one containing OG. This maybe due to high concentration of OG required I guess. When I increased concentration of protein in OG buffer, it helped increase signal-noise ratio. In this issue, I don't know if Wyatt column can help reduce the amount of sample. But I doubt if GE 5/150 column has the same resolution with the 10/30 one. When I had mixture of several species, the 10/30 always gave better resolution for me, and this is crucial in obtaining accurate information about oligomerization state and/or absolute molecular weight of the protein. So I think if choosing GE columns for light scattering experiment, I would go for 10/30. Best wishes, Sally. On Tue, Mar 8, 2011 at 7:44 PM, Engin Özkan wrote: > And as others have said shedding of dextran is a problem with GE columns > (this was confirmed to me by GE people), but after extensive system > equilibration, we do not see a problem significant enough to ever hurt our > light scattering measurements. > > Engin > > On 3/8/11 10:38 AM, Engin Özkan wrote: >> >> On 3/8/11 5:03 AM, Sebastiano Pasqualato wrote: >>> >>> On the other hand, GE Healthcare columns would require a huge amount of >>> material to be loaded. >>> >> What do you mean by a huge amount of material? You would not be using a >> 16/60 column (125 ml column volume) for an analytical experiment. How about >> a Superdex 10/300 (used to be called 10/30) or a 5/150 column. These have >> column volumes at 25 ml and 3 ml, respectively, have great resolution, and >> probably already compatible with your proteins and buffers. >> >> We used to use HPLC columns, but some proteins would never elute from >> these columns. Then we switched to good old Superdex 200 10/300, and it >> works like a charm every time. We inject <100 ul material at concentrations >> around 1 mg/ml (depending on the molecular weight of the protein in >> question). The only issue is we have to run these columns at 0.35 ml/min >> flow rates (instead of the default 0.5 ml/min), since our HPLC has a lot of >> back pressure for FPLC columns. >> >> Best, >> Engin >> > > > -- > Engin Özkan > Post-doctoral Scholar > Howard Hughes Medical Institute > Dept of Molecular and Cellular Physiology > 279 Campus Drive, Beckman Center B173 > Stanford School of Medicine > Stanford, CA 94305 > ph: (650)-498-7111 >
Re: [ccp4bb] [OT] which column to use in SLS/MALS instruments
And as others have said shedding of dextran is a problem with GE columns (this was confirmed to me by GE people), but after extensive system equilibration, we do not see a problem significant enough to ever hurt our light scattering measurements. Engin On 3/8/11 10:38 AM, Engin Özkan wrote: On 3/8/11 5:03 AM, Sebastiano Pasqualato wrote: On the other hand, GE Healthcare columns would require a huge amount of material to be loaded. What do you mean by a huge amount of material? You would not be using a 16/60 column (125 ml column volume) for an analytical experiment. How about a Superdex 10/300 (used to be called 10/30) or a 5/150 column. These have column volumes at 25 ml and 3 ml, respectively, have great resolution, and probably already compatible with your proteins and buffers. We used to use HPLC columns, but some proteins would never elute from these columns. Then we switched to good old Superdex 200 10/300, and it works like a charm every time. We inject <100 ul material at concentrations around 1 mg/ml (depending on the molecular weight of the protein in question). The only issue is we have to run these columns at 0.35 ml/min flow rates (instead of the default 0.5 ml/min), since our HPLC has a lot of back pressure for FPLC columns. Best, Engin -- Engin Özkan Post-doctoral Scholar Howard Hughes Medical Institute Dept of Molecular and Cellular Physiology 279 Campus Drive, Beckman Center B173 Stanford School of Medicine Stanford, CA 94305 ph: (650)-498-7111
Re: [ccp4bb] [OT] which column to use in SLS/MALS instruments
they buy it from some small company, idea being the applicability to SLS, optimized for minimized bleeding/sheding or material from the column, which will show up only in the light scattering detector. I don't know. Every single spec of Wyatts columns looks exactly the same as "Bio SEC-5" series of columns available from Agilent. Dima
Re: [ccp4bb] [OT] which column to use in SLS/MALS instruments
On 3/8/11 5:03 AM, Sebastiano Pasqualato wrote: On the other hand, GE Healthcare columns would require a huge amount of material to be loaded. What do you mean by a huge amount of material? You would not be using a 16/60 column (125 ml column volume) for an analytical experiment. How about a Superdex 10/300 (used to be called 10/30) or a 5/150 column. These have column volumes at 25 ml and 3 ml, respectively, have great resolution, and probably already compatible with your proteins and buffers. We used to use HPLC columns, but some proteins would never elute from these columns. Then we switched to good old Superdex 200 10/300, and it works like a charm every time. We inject <100 ul material at concentrations around 1 mg/ml (depending on the molecular weight of the protein in question). The only issue is we have to run these columns at 0.35 ml/min flow rates (instead of the default 0.5 ml/min), since our HPLC has a lot of back pressure for FPLC columns. Best, Engin -- Engin Özkan Post-doctoral Scholar Howard Hughes Medical Institute Dept of Molecular and Cellular Physiology 279 Campus Drive, Beckman Center B173 Stanford School of Medicine Stanford, CA 94305 ph: (650)-498-7111
Re: [ccp4bb] [OT] which column to use in SLS/MALS instruments
Hi, they buy it from some small company, idea being the applicability to SLS, optimized for minimized bleeding/sheding or material from the column, which will show up only in the light scattering detector. but i have to say some of our proteins stick to their columns (happened during the demo), whereas they dontt to TOSOH, on the other hand some proteins, as mentioned dont like silica at all --- so you have to try, or if you know GE columns work for you (i have some proteins that like to stick to sacharide based things...) can go with them quite ok. Few tens of micrograms is certainly enough on a 10/300 S200 for instance... narrow bore silica columns (eg 4.8 mm TOSOH) appear to be bit limiting since the flow rate would be better to be bit faster than what you can get there (<0.4 ml/min vs 0.5-1ml/min on S200 superdex) Tommi On Mar 8, 2011, at 6:56 PM, Jacob Keller wrote: So from where does Wyatt outsource their columns? JPK On Tue, Mar 8, 2011 at 10:49 AM, Sally Pham Thanh Van wrote: I should say clearer that I did a comparison between GE (Superdex 200 10/30) and the Wyatt column of the same separation range on AKTA connected with MALS. The Wyatt column gave better resolution for size exclusion chromatography as well as signal-noise ratio for light scattering. So I think it is worth for you to try it. They offer money-back guarantee. Best wishes, Sally. On Tue, Mar 8, 2011 at 3:36 PM, Sally Pham Thanh Van wrote: Dear Sebastiano, From my experiences, Wyatt columns are the best. Best wishes, Sally. On Tue, Mar 8, 2011 at 2:03 PM, Sebastiano Pasqualato wrote: Dear all, I was wondering if somebody could help me out by suggesting the "best" column to be used in a Static Light Scattering (I guess it would be the same for a Multi Angle Light Scattering) instrument. We were suggested using a silica-based column, with very high separation properties, but it seems that these columns are highly sensitive to (even slightly) basic pH's. Even running the column in PBS, it looks like injecting samples at pH 8.0 ruins the column resin, making it unusable. On the other hand, GE Healthcare columns would require a huge amount of material to be loaded. What are you guys using in your instruments? Thanks a lot in advance for the feedback, best, ciao Sebastiano -- Sebastiano Pasqualato, PhD Crystallography Unit IFOM-IEO Campus Cogentech - Consortium for Genomic Technologies via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5172 fax +39 02 9437 5990 -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** Tommi Kajander, Ph.D. Structural Biology and Biophysics Institute of Biotechnology University of Helsinki Viikinkaari 1 (P.O. Box 65) 00014 Helsinki Finland p. +358-9-19158903 tommi.kajan...@helsinki.fi
Re: [ccp4bb] [OT] which column to use in SLS/MALS instruments
So from where does Wyatt outsource their columns? JPK On Tue, Mar 8, 2011 at 10:49 AM, Sally Pham Thanh Van wrote: > I should say clearer that I did a comparison between GE (Superdex 200 > 10/30) and the Wyatt column of the same separation range on AKTA > connected with MALS. The Wyatt column gave better resolution for size > exclusion chromatography as well as signal-noise ratio for light > scattering. So I think it is worth for you to try it. They offer > money-back guarantee. > > Best wishes, > Sally. > > On Tue, Mar 8, 2011 at 3:36 PM, Sally Pham Thanh Van > wrote: >> Dear Sebastiano, >> >> From my experiences, Wyatt columns are the best. >> >> Best wishes, >> Sally. >> >> On Tue, Mar 8, 2011 at 2:03 PM, Sebastiano Pasqualato >> wrote: >>> Dear all, >>> I was wondering if somebody could help me out by suggesting the "best" >>> column to be used in a Static Light Scattering (I guess it would be the >>> same for a Multi Angle Light Scattering) instrument. >>> We were suggested using a silica-based column, with very high separation >>> properties, but it seems that these columns are highly sensitive to (even >>> slightly) basic pH's. Even running the column in PBS, it looks like >>> injecting samples at pH 8.0 ruins the column resin, making it unusable. >>> On the other hand, GE Healthcare columns would require a huge amount of >>> material to be loaded. >>> What are you guys using in your instruments? >>> Thanks a lot in advance for the feedback, >>> best, >>> ciao >>> Sebastiano >>> >>> -- >>> Sebastiano Pasqualato, PhD >>> Crystallography Unit >>> IFOM-IEO Campus >>> Cogentech - Consortium for Genomic Technologies >>> via Adamello, 16 >>> 20139 - Milano >>> Italy >>> >>> tel +39 02 9437 5172 >>> fax +39 02 9437 5990 >>> >> > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] [OT] which column to use in SLS/MALS instruments
I should say clearer that I did a comparison between GE (Superdex 200 10/30) and the Wyatt column of the same separation range on AKTA connected with MALS. The Wyatt column gave better resolution for size exclusion chromatography as well as signal-noise ratio for light scattering. So I think it is worth for you to try it. They offer money-back guarantee. Best wishes, Sally. On Tue, Mar 8, 2011 at 3:36 PM, Sally Pham Thanh Van wrote: > Dear Sebastiano, > > From my experiences, Wyatt columns are the best. > > Best wishes, > Sally. > > On Tue, Mar 8, 2011 at 2:03 PM, Sebastiano Pasqualato > wrote: >> Dear all, >> I was wondering if somebody could help me out by suggesting the "best" >> column to be used in a Static Light Scattering (I guess it would be the same >> for a Multi Angle Light Scattering) instrument. >> We were suggested using a silica-based column, with very high separation >> properties, but it seems that these columns are highly sensitive to (even >> slightly) basic pH's. Even running the column in PBS, it looks like >> injecting samples at pH 8.0 ruins the column resin, making it unusable. >> On the other hand, GE Healthcare columns would require a huge amount of >> material to be loaded. >> What are you guys using in your instruments? >> Thanks a lot in advance for the feedback, >> best, >> ciao >> Sebastiano >> >> -- >> Sebastiano Pasqualato, PhD >> Crystallography Unit >> IFOM-IEO Campus >> Cogentech - Consortium for Genomic Technologies >> via Adamello, 16 >> 20139 - Milano >> Italy >> >> tel +39 02 9437 5172 >> fax +39 02 9437 5990 >> >
Re: [ccp4bb] [OT] which column to use in SLS/MALS instruments
Hi Sebastiano, we have used both silica based and superdex columns and we found the second as a better option for general usage. Most of our proteins are on basic buffer and about 50% of them stick on the silica based column. On the other side a superdex 200 10/30 from GE performed quite well, we got quite accurate results for both a ~250kDa hexameric enzyme as also for a 0.3 mg/ml injection of an insulin sample. Eventually I think depends on the applications you prefer to have. cheers Nikos Dr Nikos Pinotsis Max F. Perutz Laboratories University of Vienna Campus Vienna Biocenter 5 A-1030 Vienna Austria Phone: +43-1-4277-52224 On 03/08/2011 02:03 PM, Sebastiano Pasqualato wrote: Dear all, I was wondering if somebody could help me out by suggesting the "best" column to be used in a Static Light Scattering (I guess it would be the same for a Multi Angle Light Scattering) instrument. We were suggested using a silica-based column, with very high separation properties, but it seems that these columns are highly sensitive to (even slightly) basic pH's. Even running the column in PBS, it looks like injecting samples at pH 8.0 ruins the column resin, making it unusable. On the other hand, GE Healthcare columns would require a huge amount of material to be loaded. What are you guys using in your instruments? Thanks a lot in advance for the feedback, best, ciao Sebastiano
Re: [ccp4bb] [OT] which column to use in SLS/MALS instruments
Hi Sebastiano, we are very happy with the SEC columns from Wyatt coupled to our FPLC/MALLS machine. http://www.wyatt.com/solutions/hardware/sec-columns-for-multi-angle-light-sc attering.html The separation range is (more or less) comparable to GE standard spdx 200/75/superose 6, and the columns are rock solid. The quality of separation is excellent in analytical runs, we routinely use them loading sample volumes of 10-25 microliters at concentrations around 1mg/ml. We did not encounter reproducibility problems/column damage so far with buffers at pH in the 6-8.5 range (8.5 is actually the high pH limit recommended by Wyatt). HTH Best, Federico Forneris, PhD --- Crystal and Structural Chemistry Bijvoet Center for Biomolecular Research - Utrecht University Padualaan 8 3584 CH UTRECHT - The Netherlands Tel. +31 (0) 30 253 2868 - Fax +31 (0) 30 253 3940 http://www.crystal.chem.uu.nl/group-gros/ -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sebastiano Pasqualato Sent: martedì 8 marzo 2011 14:04 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] [OT] which column to use in SLS/MALS instruments Dear all, I was wondering if somebody could help me out by suggesting the "best" column to be used in a Static Light Scattering (I guess it would be the same for a Multi Angle Light Scattering) instrument. We were suggested using a silica-based column, with very high separation properties, but it seems that these columns are highly sensitive to (even slightly) basic pH's. Even running the column in PBS, it looks like injecting samples at pH 8.0 ruins the column resin, making it unusable. On the other hand, GE Healthcare columns would require a huge amount of material to be loaded. What are you guys using in your instruments? Thanks a lot in advance for the feedback, best, ciao Sebastiano -- Sebastiano Pasqualato, PhD Crystallography Unit IFOM-IEO Campus Cogentech - Consortium for Genomic Technologies via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5172 fax +39 02 9437 5990
Re: [ccp4bb] [OT] which column to use in SLS/MALS instruments
Dear Sebastiano, >From my experiences, Wyatt columns are the best. Best wishes, Sally. On Tue, Mar 8, 2011 at 2:03 PM, Sebastiano Pasqualato wrote: > Dear all, > I was wondering if somebody could help me out by suggesting the "best" column > to be used in a Static Light Scattering (I guess it would be the same for a > Multi Angle Light Scattering) instrument. > We were suggested using a silica-based column, with very high separation > properties, but it seems that these columns are highly sensitive to (even > slightly) basic pH's. Even running the column in PBS, it looks like injecting > samples at pH 8.0 ruins the column resin, making it unusable. > On the other hand, GE Healthcare columns would require a huge amount of > material to be loaded. > What are you guys using in your instruments? > Thanks a lot in advance for the feedback, > best, > ciao > Sebastiano > > -- > Sebastiano Pasqualato, PhD > Crystallography Unit > IFOM-IEO Campus > Cogentech - Consortium for Genomic Technologies > via Adamello, 16 > 20139 - Milano > Italy > > tel +39 02 9437 5172 > fax +39 02 9437 5990 >
[ccp4bb] [OT] which column to use in SLS/MALS instruments
Dear all, I was wondering if somebody could help me out by suggesting the "best" column to be used in a Static Light Scattering (I guess it would be the same for a Multi Angle Light Scattering) instrument. We were suggested using a silica-based column, with very high separation properties, but it seems that these columns are highly sensitive to (even slightly) basic pH's. Even running the column in PBS, it looks like injecting samples at pH 8.0 ruins the column resin, making it unusable. On the other hand, GE Healthcare columns would require a huge amount of material to be loaded. What are you guys using in your instruments? Thanks a lot in advance for the feedback, best, ciao Sebastiano -- Sebastiano Pasqualato, PhD Crystallography Unit IFOM-IEO Campus Cogentech - Consortium for Genomic Technologies via Adamello, 16 20139 - Milano Italy tel +39 02 9437 5172 fax +39 02 9437 5990
