Re: [ccp4bb] Crystal gel band
Thanks all of you for your answers. As you guessed I was doing coomassie staining from single crystal (0.10-0.13-0.05).Fortunately I had lots of not so great looking single crystals from similar drops and I took about 10-12 of them.Then I could see a faint band where I was expecting! Thanks again. Ivan On Tue, Nov 2, 2010 at 9:15 AM, Kenneth Verstraete < kenneth.verstra...@ugent.be> wrote: > Hi Ivan, > > there are several tests (e.g. Izit dye, crush test) you can do discern > protein from salt crystals but what was always very informative to me (and > certainly in the case of complexes) is a silver-stained SDS-PAGE gel of the > crystals using the following protocol: > > - select a drop which contains some substantial crystalline material. The > crystals can be many and small (crystal shower) or few and large. > - prepare a PCR-tube with eg. 50 microliter stabilizing buffer (mother > liquor containing a 10% higher concentration of precipitant) > - transfer all the crystalline material from the drop into the PCR-tube > using a pipet (use stabilizing buffer from the PCR tube to collect all > crystals) > - centrifuge the PCR-tube at low speed for 30-60 sec and observe the > crystals under the microscope. They should be at the bottom of the PCR-tube. > - Remove as much as supernatant as you can (make sure not to remove your > crystals), add stabilizing buffer to wash the crystals, and centrifuge again > - repeat this washing protocol a few times > - after the final washing step, add Laemli-buffer to the crystals and use > this sample to load the SDS-PAGE gel > - include a positive (eg. solubilize another drop directly in > Laemli-buffer) and a negative (final washing buffer) control > - use silver staining to visualize the protein > > This always work for me. If you don't see a band a this point I would be > worried that it is salt. You could then choose to do a Western blot in stead > of silver staining to increase the sensitivity. Make your to include control > samples then. > > Kind regards, > > Kenneth Verstraete > L-PROBE > Ghent University > Belgium > > > Citeren "xaravich ivan" : > > > Hi everyone, >> I have grown some crystals after micro-seeding starting from thin-small >> needles from needle-clusters. These crystals are larger in size than the >> needles but are comparable to the shape and don't look like salt crystals. >> But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a >> home source,handy and would like to send these to the synchrotron. >> >> Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, >> the amount of protein is < 1uG? >> Has anyone experienced such a thing (no band in gel, but crystal >> diffracts)? >> >> It would be nice if I get observations/suggestions. >> >> ivan >> >> > > >
Re: [ccp4bb] Crystal gel band
Hi Ivan, there are several tests (e.g. Izit dye, crush test) you can do discern protein from salt crystals but what was always very informative to me (and certainly in the case of complexes) is a silver-stained SDS-PAGE gel of the crystals using the following protocol: - select a drop which contains some substantial crystalline material. The crystals can be many and small (crystal shower) or few and large. - prepare a PCR-tube with eg. 50 microliter stabilizing buffer (mother liquor containing a 10% higher concentration of precipitant) - transfer all the crystalline material from the drop into the PCR-tube using a pipet (use stabilizing buffer from the PCR tube to collect all crystals) - centrifuge the PCR-tube at low speed for 30-60 sec and observe the crystals under the microscope. They should be at the bottom of the PCR-tube. - Remove as much as supernatant as you can (make sure not to remove your crystals), add stabilizing buffer to wash the crystals, and centrifuge again - repeat this washing protocol a few times - after the final washing step, add Laemli-buffer to the crystals and use this sample to load the SDS-PAGE gel - include a positive (eg. solubilize another drop directly in Laemli-buffer) and a negative (final washing buffer) control - use silver staining to visualize the protein This always work for me. If you don't see a band a this point I would be worried that it is salt. You could then choose to do a Western blot in stead of silver staining to increase the sensitivity. Make your to include control samples then. Kind regards, Kenneth Verstraete L-PROBE Ghent University Belgium Citeren "xaravich ivan" : Hi everyone, I have grown some crystals after micro-seeding starting from thin-small needles from needle-clusters. These crystals are larger in size than the needles but are comparable to the shape and don't look like salt crystals. But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a home source,handy and would like to send these to the synchrotron. Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, the amount of protein is < 1uG? Has anyone experienced such a thing (no band in gel, but crystal diffracts)? It would be nice if I get observations/suggestions. ivan
Re: [ccp4bb] Crystal gel band
I recently ran mass spec analysis on some crystals that I had obtained from an optimization screen. I was looking for modifications in the protein. In order to get enough signal, I had to harvest and dissolve about 8 crystals roughly 0.3 x 0.15 x 0.15mm into the MS loading buffer in order to get a strong enough signal for the experiment. Alas, I did not find the modification I had been looking for, but I am not surprised that one single crystal, or even a small cluster of them did not show a band on a coomassie-stained gel. Do you A) have a MS handy, and B) can you sacrifice ~8 crystals to get enough S/N for a good experiment? Good luck! From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jim Pflugrath Sent: Tuesday, November 02, 2010 10:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Crystal gel band It reads like you need to run a lane or two with a positive control of some kind. Can you grow lysozyme, glucose isomerase, hemoglobin or other crystals of a protein around the same expected molecular weight and try run on the gel lanes with about the same amount of crystalline volume as your putative protein crystals? From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of xaravich ivan Sent: Monday, November 01, 2010 9:51 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystal gel band Hi everyone, I have grown some crystals after micro-seeding starting from thin-small needles from needle-clusters. These crystals are larger in size than the needles but are comparable to the shape and don't look like salt crystals. But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a home source,handy and would like to send these to the synchrotron. Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, the amount of protein is < 1uG? Has anyone experienced such a thing (no band in gel, but crystal diffracts)? It would be nice if I get observations/suggestions. ivan -- Confidentiality Notice: This message is private and may contain confidential and proprietary information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
Re: [ccp4bb] Crystal gel band
The other thing you can try to do is "the-cheap-men-silver-stain" scan your gel and bump up the contrast, you'd be surprised what you can detect even from a regular stained gel. Best results are if you make the gel first as a greyscale image. Google for Neuhoff stain and bump up the phosphoric acid to 10%. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Nov 2, 2010, at 10:19 AM, Jim Pflugrath wrote: > It reads like you need to run a lane or two with a positive control of some > kind. Can you grow lysozyme, glucose isomerase, hemoglobin or other crystals > of a protein around the same expected molecular weight and try run on the gel > lanes with about the same amount of crystalline volume as your putative > protein crystals? > > From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of > xaravich ivan > Sent: Monday, November 01, 2010 9:51 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: [ccp4bb] Crystal gel band > > Hi everyone, > I have grown some crystals after micro-seeding starting from thin-small > needles from needle-clusters. These crystals are larger in size than the > needles but are comparable to the shape and don't look like salt crystals. > But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a > home source,handy and would like to send these to the synchrotron. > > Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, > the amount of protein is < 1uG? > Has anyone experienced such a thing (no band in gel, but crystal diffracts)? > It would be nice if I get observations/suggestions. > > ivan
Re: [ccp4bb] Crystal gel band
It reads like you need to run a lane or two with a positive control of some kind. Can you grow lysozyme, glucose isomerase, hemoglobin or other crystals of a protein around the same expected molecular weight and try run on the gel lanes with about the same amount of crystalline volume as your putative protein crystals? _ From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of xaravich ivan Sent: Monday, November 01, 2010 9:51 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Crystal gel band Hi everyone, I have grown some crystals after micro-seeding starting from thin-small needles from needle-clusters. These crystals are larger in size than the needles but are comparable to the shape and don't look like salt crystals. But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a home source,handy and would like to send these to the synchrotron. Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, the amount of protein is < 1uG? Has anyone experienced such a thing (no band in gel, but crystal diffracts)? It would be nice if I get observations/suggestions. ivan
Re: [ccp4bb] Crystal gel band
I have grown some crystals after micro-seeding starting from thin-small
needles from needle-clusters. These crystals are larger in size than the
needles but are comparable to the shape and don't look like salt crystals.
But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a
home source,handy and would like to send these to the synchrotron.
Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say,
the amount of protein is < 1uG?
Protein to protein variation aside (which is usually within 2X),
conventional Coomassie R-250 staining gives very-easy-no-doubt detection
down to ~0.1 ug. 50 ng is still visible (assuming that the gel is any
good). Colloidal Coomassie G-250 ("Bradford") with destaining extends the
range down to 30-20 ng or 10 ng with some effort and special staining
solution. Proper silver staining should easily see 1 ng. I.e., a single
crystal that is a 50 um cube with 50% solvent puts you in the borderline
zone of "easy". Load many of them to be sure or use silver.
- Dima
Re: [ccp4bb] Crystal gel band
Hi ivan, The detection senstivity of proteins depends on staining technique, You have not mentioned about staining, whether silver or Coomassie. The silver staining is more sensitive than coomassie, typically you can see 10-50 ng of a protein in silver staining. It does vary, however, with the glycosylation and physical properties of the protein. Shivendra On 2 November 2010 08:20, xaravich ivan wrote: > Hi everyone, > I have grown some crystals after micro-seeding starting from thin-small > needles from needle-clusters. These crystals are larger in size than the > needles but are comparable to the shape and don't look like salt crystals. > But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a > home source,handy and would like to send these to the synchrotron. > > Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, > the amount of protein is < 1uG? > Has anyone experienced such a thing (no band in gel, but crystal > diffracts)? > It would be nice if I get observations/suggestions. > > ivan >
[ccp4bb] Crystal gel band
Hi everyone, I have grown some crystals after micro-seeding starting from thin-small needles from needle-clusters. These crystals are larger in size than the needles but are comparable to the shape and don't look like salt crystals. But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a home source,handy and would like to send these to the synchrotron. Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, the amount of protein is < 1uG? Has anyone experienced such a thing (no band in gel, but crystal diffracts)? It would be nice if I get observations/suggestions. ivan
