Re: [ccp4bb] off topic: good peak on gel filtration
OK, lets see- 25000 plates/m = 7500 plates in a 30 cm column, so variance in elution volume of the molecules should be elution volume/sqrt(7500) = .0115 Ve. Full width at half height is 2*sqrt(2ln2), or 2.35, times sigma, so 0.0275 times elution volume. For elution volumes 10 - 15 ml, FWHH of peak should be 0.27 to 0.4 ml, if the sample is applied in an infinitely small volume. For finite samples I guess width of the peak would be the sample volume increased by .27 to .4 ml due to peak broadening. eab Zhijie Li wrote: Hi Ed, I guess by 24mL SD200 Peter meant the Superdex 200 10/300 column, which most of us should be quite familiar with. According to GE healthcare, a new Superdex 200 10/300 GL column should have TP 25000/m. For comparison, a new Superdex200 16/600 PG, which uses bigger beads, has TP 13000/m. The TP difference of the two should be mainly caused by the different resin sizes. Of course in reality columns change over time and in cases like Peter's, it might be a good idea to test the performance of the column before drawing a conclusion. When we are concerned about resolution of a column, we load a standard sample and calculate the TP based on the peak shape. As I remember, GE healthcare's SEC manuals has recommended procedures on TP determination. Zhijie -Original Message- From: Edward A. Berry Sent: Saturday, June 29, 2013 7:43 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] off topic: good peak on gel filtration Peter Hsu wrote: Hi all, I've generally always thought as long as the peak was symmetrical and not too broad would suggest a good sample. However, looking at my previous runs in the past, I've had peaks as narrow as 1.5-2mL on a 24mL SD200, or slightly broader peaks with about 3mL (all symmetrical peaks, roughly similar amounts loaded on the columns). I'm curious to see what people's views are as far as what constitutes a broad peak and how much that can end up affecting crystallization of the sample. Thanks for any responses. Peter The width itself may not be a good indicator unless its always the same protein- in general a molecule that elutes later will have a broader peak. Supposing that each time a molecule diffuses into the stationary phase it resides there for a certain time on the average, then the extra retention time is proportional to that time, times the number of times it enters stationary phase (N, theoretical plates). The variance in elution time is proportional to the square root of N (like standard error of the mean) and the dwell time. This gives sigma/(retention time) = 1/sqrt(N). If N is the same for all molecules, the criterion to look at is peak width divided by retention time. If it varies (the reason some molecules elute slower is not just that they stay in the stationary phase longer, but also they enter more often; k-on as well as k-off) that would still be better than just peak width. People don't talk about theoretical plats and HTEP much any more, perhaps because the driving force in chromatography is HPLC and FPLC, and fast chromatography is antithetical to good resolution? However I'm not familiar with this column and can't advise. You can calculate N more exactly (see wikipedia van Deemter equation) as 8*ln(2)*square of (elution volume over width at half height), divide length of column by that to get HETP, and compare with values like .7 mm reported for resins like ultragel A at optimum (very slow) flow rate. eab
Re: [ccp4bb] off topic: good peak on gel filtration
In our lab we do DLS on the SEC fractions and only set up those that are monodisperse :-) On Mon, Jul 1, 2013 at 7:37 PM, El Arnaout, Toufic elarnao...@biochem.wustl.edu wrote: Hello Peter, In addition to the great comments/details, please check the following points I have now in mind.. since you want to relate the size exclusion peak/profile to the crystallization: - occasionnaly, some perfect peaks that you might think are homogeneous actually correspond to a sample of hetergeneous protein (maybe the target protein will still crystallize, but problems happen during crystal optimization, or/and observing missing electronic density of the N- or C-terminus for example). It might also be reflected on the specific activity (btw if the protein you have is easy to assay, you could check the activity from different fractions if you think broad peak = problem). In some occasions, analyzing fractions from a perfect peak shows on SDS-PAGE a double band or sometimes far bands (I won't comment on oligomerization in this case). - not all proteins from good SE chromatograms crystallize... - some people only collect fractions from the centre of the peak (or for example they measure the A280 max, divide it by two, draw an horizontal line at that value, and collect the fractions/projection between where the line crosses the peak from both sides.. If you add one fraction before or after, the protein might not crystallize anymore. - from the literature, I have seen many shapes of chromatograms: perfect, bleeding, skewed (tail) to the right or left, little broad, etc.. which resulted in diffraction quality crystals. - re-running a protein sample (that crystallizes after the first SEC) for a second SEC might cause the protein not to crystallize anymore (for example membrane proteins might lose lipids etc). - for proteins that are subjected to SEC straight after Ni-NTA, and which have a perfect peak shape: a 4-16 hours delay before injection might show the aggregation effect. A peak shoulder will form, which you might not have seen if the sample was directly injected. The point is: maybe what you incubate for crystallization or work on after SEC might not be anymore that protein with the beautiful peak you had. Using different additives/chemicals/mutations may help in making the protein more stable/thermostable. You can check previous publications of Dr Tate CG, GPCRs for example. This is also a nice paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3111809. Regards toufic el arnaout School of Medicine - 660 S Euclid Ave Washington University in St. Louis St Louis, MO 63110, USA From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Edward A. Berry [ber...@upstate.edu] Sent: Saturday, June 29, 2013 9:34 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] off topic: good peak on gel filtration Thanks- guess I'm old-fashioned, using low-pressure columns. So apparently theoretical plates are still calculated, and have improved a lot- 25000/m is HETP .04 mm, way better than the figure I mentioned. (TP per dollar not so much.) No more sour grapes from me- eab Zhijie Li wrote: Hi Ed, I guess by 24mL SD200 Peter meant the Superdex 200 10/300 column, which most of us should be quite familiar with. According to GE healthcare, a new Superdex 200 10/300 GL column should have TP 25000/m. For comparison, a new Superdex200 16/600 PG, which uses bigger beads, has TP 13000/m. The TP difference of the two should be mainly caused by the different resin sizes. Of course in reality columns change over time and in cases like Peter's, it might be a good idea to test the performance of the column before drawing a conclusion. When we are concerned about resolution of a column, we load a standard sample and calculate the TP based on the peak shape. As I remember, GE healthcare's SEC manuals has recommended procedures on TP determination. Zhijie -Original Message- From: Edward A. Berry Sent: Saturday, June 29, 2013 7:43 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] off topic: good peak on gel filtration Peter Hsu wrote: Hi all, I've generally always thought as long as the peak was symmetrical and not too broad would suggest a good sample. However, looking at my previous runs in the past, I've had peaks as narrow as 1.5-2mL on a 24mL SD200, or slightly broader peaks with about 3mL (all symmetrical peaks, roughly similar amounts loaded on the columns). I'm curious to see what people's views are as far as what constitutes a broad peak and how much that can end up affecting crystallization of the sample. Thanks for any responses. Peter The width itself may not be a good indicator unless its always the same protein- in general a molecule that elutes later will have a broader peak. Supposing that each time a molecule diffuses
Re: [ccp4bb] off topic: good peak on gel filtration
Hello Peter, In addition to the great comments/details, please check the following points I have now in mind.. since you want to relate the size exclusion peak/profile to the crystallization: - occasionnaly, some perfect peaks that you might think are homogeneous actually correspond to a sample of hetergeneous protein (maybe the target protein will still crystallize, but problems happen during crystal optimization, or/and observing missing electronic density of the N- or C-terminus for example). It might also be reflected on the specific activity (btw if the protein you have is easy to assay, you could check the activity from different fractions if you think broad peak = problem). In some occasions, analyzing fractions from a perfect peak shows on SDS-PAGE a double band or sometimes far bands (I won't comment on oligomerization in this case). - not all proteins from good SE chromatograms crystallize... - some people only collect fractions from the centre of the peak (or for example they measure the A280 max, divide it by two, draw an horizontal line at that value, and collect the fractions/projection between where the line crosses the peak from both sides.. If you add one fraction before or after, the protein might not crystallize anymore. - from the literature, I have seen many shapes of chromatograms: perfect, bleeding, skewed (tail) to the right or left, little broad, etc.. which resulted in diffraction quality crystals. - re-running a protein sample (that crystallizes after the first SEC) for a second SEC might cause the protein not to crystallize anymore (for example membrane proteins might lose lipids etc). - for proteins that are subjected to SEC straight after Ni-NTA, and which have a perfect peak shape: a 4-16 hours delay before injection might show the aggregation effect. A peak shoulder will form, which you might not have seen if the sample was directly injected. The point is: maybe what you incubate for crystallization or work on after SEC might not be anymore that protein with the beautiful peak you had. Using different additives/chemicals/mutations may help in making the protein more stable/thermostable. You can check previous publications of Dr Tate CG, GPCRs for example. This is also a nice paper: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3111809. Regards toufic el arnaout School of Medicine - 660 S Euclid Ave Washington University in St. Louis St Louis, MO 63110, USA From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Edward A. Berry [ber...@upstate.edu] Sent: Saturday, June 29, 2013 9:34 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] off topic: good peak on gel filtration Thanks- guess I'm old-fashioned, using low-pressure columns. So apparently theoretical plates are still calculated, and have improved a lot- 25000/m is HETP .04 mm, way better than the figure I mentioned. (TP per dollar not so much.) No more sour grapes from me- eab Zhijie Li wrote: Hi Ed, I guess by 24mL SD200 Peter meant the Superdex 200 10/300 column, which most of us should be quite familiar with. According to GE healthcare, a new Superdex 200 10/300 GL column should have TP 25000/m. For comparison, a new Superdex200 16/600 PG, which uses bigger beads, has TP 13000/m. The TP difference of the two should be mainly caused by the different resin sizes. Of course in reality columns change over time and in cases like Peter's, it might be a good idea to test the performance of the column before drawing a conclusion. When we are concerned about resolution of a column, we load a standard sample and calculate the TP based on the peak shape. As I remember, GE healthcare's SEC manuals has recommended procedures on TP determination. Zhijie -Original Message- From: Edward A. Berry Sent: Saturday, June 29, 2013 7:43 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] off topic: good peak on gel filtration Peter Hsu wrote: Hi all, I've generally always thought as long as the peak was symmetrical and not too broad would suggest a good sample. However, looking at my previous runs in the past, I've had peaks as narrow as 1.5-2mL on a 24mL SD200, or slightly broader peaks with about 3mL (all symmetrical peaks, roughly similar amounts loaded on the columns). I'm curious to see what people's views are as far as what constitutes a broad peak and how much that can end up affecting crystallization of the sample. Thanks for any responses. Peter The width itself may not be a good indicator unless its always the same protein- in general a molecule that elutes later will have a broader peak. Supposing that each time a molecule diffuses into the stationary phase it resides there for a certain time on the average, then the extra retention time is proportional to that time, times the number of times it enters stationary phase (N, theoretical plates). The variance in elution time is proportional
Re: [ccp4bb] off topic: good peak on gel filtration
Peter Hsu wrote: Hi all, I've generally always thought as long as the peak was symmetrical and not too broad would suggest a good sample. However, looking at my previous runs in the past, I've had peaks as narrow as 1.5-2mL on a 24mL SD200, or slightly broader peaks with about 3mL (all symmetrical peaks, roughly similar amounts loaded on the columns). I'm curious to see what people's views are as far as what constitutes a broad peak and how much that can end up affecting crystallization of the sample. Thanks for any responses. Peter The width itself may not be a good indicator unless its always the same protein- in general a molecule that elutes later will have a broader peak. Supposing that each time a molecule diffuses into the stationary phase it resides there for a certain time on the average, then the extra retention time is proportional to that time, times the number of times it enters stationary phase (N, theoretical plates). The variance in elution time is proportional to the square root of N (like standard error of the mean) and the dwell time. This gives sigma/(retention time) = 1/sqrt(N). If N is the same for all molecules, the criterion to look at is peak width divided by retention time. If it varies (the reason some molecules elute slower is not just that they stay in the stationary phase longer, but also they enter more often; k-on as well as k-off) that would still be better than just peak width. People don't talk about theoretical plats and HTEP much any more, perhaps because the driving force in chromatography is HPLC and FPLC, and fast chromatography is antithetical to good resolution? However I'm not familiar with this column and can't advise. You can calculate N more exactly (see wikipedia van Deemter equation) as 8*ln(2)*square of (elution volume over width at half height), divide length of column by that to get HETP, and compare with values like .7 mm reported for resins like ultragel A at optimum (very slow) flow rate. eab
Re: [ccp4bb] off topic: good peak on gel filtration
Hi Ed, I guess by 24mL SD200 Peter meant the Superdex 200 10/300 column, which most of us should be quite familiar with. According to GE healthcare, a new Superdex 200 10/300 GL column should have TP 25000/m. For comparison, a new Superdex200 16/600 PG, which uses bigger beads, has TP 13000/m. The TP difference of the two should be mainly caused by the different resin sizes. Of course in reality columns change over time and in cases like Peter's, it might be a good idea to test the performance of the column before drawing a conclusion. When we are concerned about resolution of a column, we load a standard sample and calculate the TP based on the peak shape. As I remember, GE healthcare's SEC manuals has recommended procedures on TP determination. Zhijie -Original Message- From: Edward A. Berry Sent: Saturday, June 29, 2013 7:43 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] off topic: good peak on gel filtration Peter Hsu wrote: Hi all, I've generally always thought as long as the peak was symmetrical and not too broad would suggest a good sample. However, looking at my previous runs in the past, I've had peaks as narrow as 1.5-2mL on a 24mL SD200, or slightly broader peaks with about 3mL (all symmetrical peaks, roughly similar amounts loaded on the columns). I'm curious to see what people's views are as far as what constitutes a broad peak and how much that can end up affecting crystallization of the sample. Thanks for any responses. Peter The width itself may not be a good indicator unless its always the same protein- in general a molecule that elutes later will have a broader peak. Supposing that each time a molecule diffuses into the stationary phase it resides there for a certain time on the average, then the extra retention time is proportional to that time, times the number of times it enters stationary phase (N, theoretical plates). The variance in elution time is proportional to the square root of N (like standard error of the mean) and the dwell time. This gives sigma/(retention time) = 1/sqrt(N). If N is the same for all molecules, the criterion to look at is peak width divided by retention time. If it varies (the reason some molecules elute slower is not just that they stay in the stationary phase longer, but also they enter more often; k-on as well as k-off) that would still be better than just peak width. People don't talk about theoretical plats and HTEP much any more, perhaps because the driving force in chromatography is HPLC and FPLC, and fast chromatography is antithetical to good resolution? However I'm not familiar with this column and can't advise. You can calculate N more exactly (see wikipedia van Deemter equation) as 8*ln(2)*square of (elution volume over width at half height), divide length of column by that to get HETP, and compare with values like .7 mm reported for resins like ultragel A at optimum (very slow) flow rate. eab
Re: [ccp4bb] off topic: good peak on gel filtration
Hi Peter, I won't claim to be an expert, however, there is a small textbook by Robert K. Scopes called Protein Purification: Principles and Practice, that has very good practical and theoretical information about column chromatography (among other methods). I have found this book useful a number of times when I have had similar questions. Good luck, Mike - Original Message - From: Peter Hsu hsuu...@u.washington.edu To: CCP4BB@JISCMAIL.AC.UK Sent: Saturday, June 29, 2013 2:01:15 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] off topic: good peak on gel filtration Hi all, I've generally always thought as long as the peak was symmetrical and not too broad would suggest a good sample. However, looking at my previous runs in the past, I've had peaks as narrow as 1.5-2mL on a 24mL SD200, or slightly broader peaks with about 3mL (all symmetrical peaks, roughly similar amounts loaded on the columns). I'm curious to see what people's views are as far as what constitutes a broad peak and how much that can end up affecting crystallization of the sample. Thanks for any responses. Peter -- Michael C. Thompson Graduate Student Biochemistry Molecular Biology Division Department of Chemistry Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] off topic: good peak on gel filtration
Thanks- guess I'm old-fashioned, using low-pressure columns. So apparently theoretical plates are still calculated, and have improved a lot- 25000/m is HETP .04 mm, way better than the figure I mentioned. (TP per dollar not so much.) No more sour grapes from me- eab Zhijie Li wrote: Hi Ed, I guess by 24mL SD200 Peter meant the Superdex 200 10/300 column, which most of us should be quite familiar with. According to GE healthcare, a new Superdex 200 10/300 GL column should have TP 25000/m. For comparison, a new Superdex200 16/600 PG, which uses bigger beads, has TP 13000/m. The TP difference of the two should be mainly caused by the different resin sizes. Of course in reality columns change over time and in cases like Peter's, it might be a good idea to test the performance of the column before drawing a conclusion. When we are concerned about resolution of a column, we load a standard sample and calculate the TP based on the peak shape. As I remember, GE healthcare's SEC manuals has recommended procedures on TP determination. Zhijie -Original Message- From: Edward A. Berry Sent: Saturday, June 29, 2013 7:43 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] off topic: good peak on gel filtration Peter Hsu wrote: Hi all, I've generally always thought as long as the peak was symmetrical and not too broad would suggest a good sample. However, looking at my previous runs in the past, I've had peaks as narrow as 1.5-2mL on a 24mL SD200, or slightly broader peaks with about 3mL (all symmetrical peaks, roughly similar amounts loaded on the columns). I'm curious to see what people's views are as far as what constitutes a broad peak and how much that can end up affecting crystallization of the sample. Thanks for any responses. Peter The width itself may not be a good indicator unless its always the same protein- in general a molecule that elutes later will have a broader peak. Supposing that each time a molecule diffuses into the stationary phase it resides there for a certain time on the average, then the extra retention time is proportional to that time, times the number of times it enters stationary phase (N, theoretical plates). The variance in elution time is proportional to the square root of N (like standard error of the mean) and the dwell time. This gives sigma/(retention time) = 1/sqrt(N). If N is the same for all molecules, the criterion to look at is peak width divided by retention time. If it varies (the reason some molecules elute slower is not just that they stay in the stationary phase longer, but also they enter more often; k-on as well as k-off) that would still be better than just peak width. People don't talk about theoretical plats and HTEP much any more, perhaps because the driving force in chromatography is HPLC and FPLC, and fast chromatography is antithetical to good resolution? However I'm not familiar with this column and can't advise. You can calculate N more exactly (see wikipedia van Deemter equation) as 8*ln(2)*square of (elution volume over width at half height), divide length of column by that to get HETP, and compare with values like .7 mm reported for resins like ultragel A at optimum (very slow) flow rate. eab
