[RASMB] Can DCDT+ be used to detect small change in protein conformation? What is its limit?

[Walter Stafford]

Hi Chris,

	Yes, DCDT can be used to detect very small changes in s. If 
you match the menisi using a capillary type synthetic boundary cell 
so the the sample on each side starts sedimenting at the same 
position. You can iether compare two samples directlyIf you are 
adding a low molecular weight ligand, you must put the sample plus 
ligand on the "sample" side , fill the "reference" side slightly more 
(20uL) than the sample side with sample without ligand. Take the run 
up to 3-10K rpm to match the menisci. Then stop the run, gently shake 
the rotor to obtain a uniform distribution and restart. A small 
amount of sample containing no ligand (10uL) will flow in to the 
sample and cause a very small dilution of ligand. The sample must 
have been dialyzed vs. the basic buffer without ligand before being 
divided. When you add ligand to the sample also add an equal amount 
of buffer to sample not containing buffer to dilute it by the same 
amount. The ligand should be dissolved in the dialysate. Am assuming 
that you are using the interference optics. The data can then be 
analyzed with DCDT. by fitting the boundary to two gaussians - on 
positive and one negative. I will give all the details if you contact 
me directly. You can also fit the data using numerical solutions to 
the Lamm equation using SedAnal.

Walter Stafford

At 13:55 -0500 9/20/02, Chin, Christopher wrote:
>To John, all DCDT+ users and all interested colleagues,
>
>  John, I would like to have your comment in the following three 
>experiments using RNAseA as a model and DCDT+ as the method to do 
>data analysis.
>
>Experiment 1:  The purpose is to check the stability of RNAseA. The 
>comparison is made between the protein left at room temperature 
>for a couple of days and that storage in the freezer.
>Experiment 2:  Question: Can the denatured RNaseA (has 4 s-s bond) 
>in GuHCl be refolded back to its native state in the absence of 
>reducing agent?
>Experiment 3:  Question: Can the denatured RNaseA (has 4 s-s bond) 
>in GuHCl be refolded back to its native state in the presence of 
>reducing agent  TBP (Tri-n-butylphosphine).
>
>The major issue here is how I can differentiate the DS difference 
>due to scan chosen from the DS difference due to real experimental 
>finding (between the control and the sample).
>
>I am pleased to say that, using the criteria that I have stated, 
>e.g. maximized or magnified the DS difference due to scan 
>chosen (use a smaller number for denominator when converted to 
>%), minimized or play down the DS difference between the control and 
>the sample (use a larger number for denominator when converted 
>to % ), the results are pretty much I have expected.  Do you think 
>I try to push the instrument limit too far to obtain these results 
>by manipulating the treatment of data?
>
>s20,w values reported here are the results of fitting dcdt.
>
>Thank you in advance for your comment or advice.
>
>Chris
>
>
>--------------------------------------------------------------------------
>Christopher Chin
>Manager, XLA-Analytical Ultracentrifugation facility
>Sealy Center for Structural Biology
>HBC&G, 5.134 MRB.UTMB, Galveston,TX 77555-1055
>cchin at utmb.edu, 409-772-1693, efax 708-585-1920
>---------------------------------------------------------------------------
>
>
>
>Attachment converted: WFS3HD:Stability of RNAse.doc (WDBN/MSWD) (00121490)
>Attachment converted: WFS3HD:GuHCl treated(without reducing 
>(WDBN/MSWD) (00121491)
>Attachment converted: WFS3HD:GuHCl-TBP treated and renatural 
>(WDBN/MSWD) (00121492)

-- 
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Walter F. Stafford III, Ph.D.
Senior Scientist
Analytical Ultracentrifugation Research Laboratory
Boston Biomedical Research Institute
64 Grove Street
Watertown, MA  02472-2829
and
Associate in Neurology
Massachusetts General Hospital
Harvard Medical School
Boston MA 02115

main:(617) 926-8040
tel: (617) 658-7808
fax: (617) 972-1753

mailto:stafford at bbri.org


http://www.bbri.org/faculty/stafford/Stafford.html
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