Reference:  
Bagby, S., Tong, K.I., Liu, D., Alattia, J.-R. and Ikura, M.  The button test:
a small scale method using microdialysis cells for assessing protein 
solubility at concentrations suitable for NMR.  J. Biomol. NMR 10:279-282.


PROCEDURE FOR SETTING UP THE BUTTON TEST

1.  The protein sample was first exchanged into a buffer system of lower
    ionic strength than the test buffer systems such that an osmotic gradient
    was formed from the test buffer side to the sample side.  In our screening
    of solution conditions with carboxy-terminal core domain (TFIIBc) of 
    human TFIIB, for example, we first exchanged the protein into distilled,
    deionised water containing 7.5 mM dithiothreitol (DDT).  The protein was
    subsequently concentrated to 1 mM or higher in order to simulate 
    conditions in an NMR sample.  An initial screen using protein at lower 
    concentration might be informative for particularly troublesome systems.

2.  A standard piece of dialysis membrane (for example, Spectra/Por molecular
    porous membrane from Spectrum, 6000 - 8000 molecular weight cut-off) was
    prepared according to the manufacturer's instructions.

3.  The wet dialysis membrane was cut into one inch squares and these pieces
    were kept moist by placing them between wet Kimwipes.  

4.  Using a micro pipette tip (for example, Bio-Rad tips for protein 
    electrophoresis, catalogue number 223-9915), 5 uL of concentrated protein
    sample was pipetted into the central well on the top surface of the 
    microdialysis cell (Fig. 1).  A circular motion was used to avoid air 
    bubble formation in the protein solution.

5.  The membrane was secured with a rubber O-ring which fits in the groove
    running around the circumference of the microdialysis cell.

6.  The microdialysis cell was submerged in 5 mL of test solution contained, 
    for example, within a scintillation vial.  In the case of the complex 
    between TBP core domain and residues 11-77 of TAFII230, the test solutions
    were pre-cooled to 4C and then kept at 4C for 2-4 hours after submersion 
    of the microdialysis cell to allow equilibration before transferring to a 
    higher temperature environment.

7.  For the TFIIBc screen, the test samples were placed in a temperature-
    controlled environment at 25C.  Each day, the clarity of the samples was 
    monitored using the naked eye and/or a standard dissecting microscope. 
    The effect of temperature on solubility can also be investigated by
    placing test samples with the same solution conditions at different
    temperatures.

8.  In order to confirm that any precipitation, discoloration or fibre
    formation is dependent on the presence of protein, microdialysis cells
    were also set up containing buffer solutions with no protein.   

9.  Microdialysis cells can be obtained from:

		Hampton Research, 25431 Cabot Road, Suite 205
		Laguna Hills, CA 92653-5527 U.S.A. 
		phone (714) 699-1040  fax (714) 586-1453
 		http://www.hamptonresearch.com

    The same company also makes O-ring applicators which facilitate the 
    process of securing the dialysis membrane.