Sedimentation Velocity Information

 1. Label your samples S#1, S#2, S#3, and the corresponding buffer B#1, B#2, B#3, such that your expected sedimentation coefficient is the largest for S#1 and the smallest for S#3. If you supply the same sample in three different concentrations, label the lowest concentration S#1 and the highest concentration S#3. The buffer must be an exact match of that used to prepare the sample.

2. Take a wavelength scan of B#1 against water from 200 to 300 nm. Do the same with B#2 and B#3 if you have three different buffers (Note: some buffers have been scanned already and can be looked up here). IMPORTANT: For each sample identify the components in the buffer on the chemical compatibility table to assure that the buffer components do not damage ultracentrifuge cell components. Charcoal/epoxy cell components (for runs up to 40,000 rpm) and anodized aluminum cell components (for runs between 40,000 and 60,000 rpm) MUST have satisfactory resistance (S) to the chemicals included in your buffer system. Buffer systems containing chemicals with unsatisfactory or damaging properties cannot be accepted. If you fail to inform us of such chemicals, we will hold you liable for any damages to the equipment. If your samples contain chemicals not listed in the table, we cannot process your samples.

3. Take a wavelength scan of each sample (S#1, S#2, S#3) against the buffer from 200 to 300 nm. Select a wavelength such that the absorbance of the buffer against water is less than 0.5 OD and the absorbance of the sample against its respective buffer is at least 0.7 OD, but no more than 1.3 OD. Make sure that the total absorbance of buffer and sample combined does not exceed 1.5 OD. (For example, you cannot use a sample absorbance of 1.5 OD with a buffer absorbance of 0.5 OD, because the total absorbance would be 2.0 OD). If possible, select a wavelength that measures the peak of your sample. This is especially important for equilibrium experiments.

4. We strongly urge that you spin your sample in a micro centrifuge at maximum speed for 15-20 minutes and recheck the absorbance. If the sample OD has decreased, your sample may be aggregating. This will have to be corrected before proceeding with this guide.

5. If your sample has a higher OD at this wavelength than 1.3 (against the buffer), dilute the sample. After dilution, repeat the wavelength scan. If you have two peaks in the wavelength scan for your sample, use the peak with higher absorbance, if the buffer absorbance allows it (i.e., if the combined absorbance for buffer and sample is below1.5 OD). If the sample is too dilute, you will have to either concentrate it with a centricon column or prepare a new sample.

6. Fill out the attached form with detailed descriptions for each sample, giving as much information as possible. Furnish photocopies of the wavelength scans and data. A photograph of a polyacrylamide or agarose gel of your sample is also very useful. Finally, indicate what temperature range is required for your samples.

7. Provide at least 500ul sample and 10 ml buffer for each measurement.

8. Please complete the following table of information:

 Name:

Address:

City:

State:

Zip code:

E-mail address:

Telephone:

Fax:

 

	Sample 1 (S#1)	Sample 2 (S#2)	Sample 3 (S#3)
Brief Description of Sample
Approx. Molecular Weight
Wavelength(s)  (nm)
Buffer components and concentrations
OD of Sample vs. Buffer
OD of Buffer vs. Water
Total OD Sample + Buffer
Partial Spec. Volume (V-bar)
Density of Buffer rel. to H2O at 20C
Viscosity of Buffer rel. to H2O at 20C
Run Temperature
Storage conditions (temperature)
Special Notes

Amino Acid Sequence, Sample 1:

Please enter a description for the amino acid sequence of sample 1:
 
Please enter the amino acid sequence of sample 1 (single letter code):

Amino Acid Sequence, Sample 2:

Please enter a description for the amino acid sequence of sample 2:
 
Please enter the amino acid sequence of sample 2 (single letter code):

Amino Acid Sequence, Sample 3:

Please enter a description for the amino acid sequence of sample 3:
 
Please enter the amino acid sequence of sample 3 (single letter code):

Please check:

Yes No

I have read the Chemical Compatibility Table and understand that I am liable for any damages caused by failing to identify damaging chemicals in my buffer system

Please make sure you click on the submit button below to submit your sample information to the Analytical Ultracentrifugation Facility. We will then take the appropriate measures to schedule your run.

 If you have a gel photograph of your sample(s), please send them by mail to:

CAMIS
Dept. of Biochemistry & Molecular Biology
Attn: Kari Hartman
46 College Road- Rudman Hall Rm 353

University of New Hampshire, Durham, NH 03824

 Alternatively, you can upload a tif or bmp formatted image file of your scanned gel to our server. Please send an e-mail to khartman@cisunix.unh.edu to notify us of the upload.

 Wavelength data (plot and numbers) can be faxed to (603) 862-4013 or an ascii version of OD vs. wavelength data can be sent by e-mail to khartman@cisunix.unh.edu

Kari L. Hartman
Copyright © 1999 [CAMIS]. All rights reserved.
Revised: August 16, 2000