Protocol for analytical ultracentrifugation sample preparation: 1.…

Protocol for analytical ultracentrifugation sample preparation:

1. Provide partial specific volume (v-bar) of the proteins. If v-bar is not available, send
   complete amino acid sequence of your protein via e-mail to rongmin.zhao@utoronto.ca.
   Please type the sequence into the body of the e-mail rather than sending it as an attached file.

2. Provide minimum 250 µL of each sample to be analyzed and at least 5 mL of buffer.

3. For accurate determination of molecular weight or oligomeric state, provide three (3)
   different concentrations for each sample. An example of an ideal concentration series is 0.25
   mg/mL, 0.50 mg/mL, and 1.00 mg/mL. Ensure that the absorbance at either 230 nm or 280
   nm (or, whatever wavelength desired) is not less than 0.25 at the lowest concentration, and
   does not exceed 1.00 at the highest concentration. Concentrations as low as 1 µg/mL are
   acceptable only if the absorbance at 230 nm is at least 0.30.
   Please make your own dilutions.

4. Try to avoid using DTT, 2-mercaptoethanol, and protease inhibitors. If DTT is absolutely
   required to maintain sample stability, do not exceed 5 mM concentration.

5. The reference buffer should contain all components that are in the sample minus the protein.

6. Give samples, buffers, and filled form to Rongmin Zhao.

7. Samples will be stored at 4oC and run at 20oC, unless otherwise specified.

Acceptable buffer components:
1-Propanol              2-Propanol                 Acetic Acid              Acetone
Ammonium chloride       Ammonium hydroxide         Ammonium sulfate         Barium chloride
Cadmium chloride        Cadmium sulfate            Calcium chloride         Cesium chloride
Citric acid             Cobaltous chloride         Creatinine               Cupric sulfate
EDTA disodium           Ethanol                    Ethylene glycol          Ferric chloride
Formic acid             Fructose                   Glucose                  Glycerol
Guanidine hydrochloride Hydrochloric acid          Inulin                   Lactic acid
Lactose                 Lanthanum nitrate          Lead nitrate             Lithium chloride
Magnesium chloride      Magnesium sulfate          Maltose                  Manganous sulfate
Mannitol                Methanol                   Nickel sulfate           Nitric acid
Oxalic acid             Phosphoric acid            Potassium bicarbonate    Potassiuim biphthalate
Potassium bromide       Potassium carbonate        Potassium chloride       Potassium chromate
Potassium dichromate    Potassium ferricyanide     Potassium ferrocyanide   Potassium hydroxide
Potassium iodide        Potassium nitrate          Potassium oxalate        Potassium permanganate
Potassium PO4 di-basic  Potassium PO4 mono-basic   Potassium sulfate        Potassium thiocyanate
Procaine hydrochloride  Propylene glycol           Silver nitrate           Sodium acetate
Sodium bicarbonate      Sodium bromide             Sodium carbonate         Sodium chloride
Sodium citrate          Sodium diatrizoate         Sodium dichromate        Sodium ferrocyanide
Sodium hydroxide        Sodium molybdate           Sodium nitrate           Sodium PO4 di-basic
Sodium PO4 mono-basic   Sodium PO4 tri-basic       Sodium sulfate Sodium tartrate
Sodium thiocyanate      Sodium thiosulfate         Sodium tungstate         Strontium chloride
Sucrose                 Sulfuric acid              Tartaric acid            Tetracaine hydrochloride
Trichloroacetic acid    Trifluoroethanol           Tris (hydroxymethyl)aminomethane
Urea                    Zinc sulfate

Please note that Dr. Avi Chakrabartty's lab at OCI also provides analytical ultracentrifugation
services (http://www.oci.utoronto.ca/institutes/html/oci/msb/chakrabartty.html).