User:Mar/Notebook/2007-6-15

= protein extract fractionation (cont) =

Bill fractioned each pool into 5 fractions of the same volume, buffer-exchanged by dialysis (10kDa), and pooled 2 runs. Total protein used for fractionation was 222 (#1) + 362 (#2) = 584 µg, what gives rough estimate of 584/5= 116.8 µg/fraction.

Fractions volumes:
 * 1)  init.vol. init.conc. conc'd vol.  conc.factor
 * 2) 1 1.6878g - 6.139mg/mL - 46 mg      37x
 * 3) 2 2.8736g - 6.153mg/mL - 89 mg      32x
 * 4) 3 3.0735g - 5.833mg/mL - 220 mg     14x
 * 5) 4 2.8639g - 6.070mg/mL - 76 mg      38x
 * 6) 5 3.0371g - 5.429mg/mL - 58 mg      52x

Note: the protein content in the fractions is unexplainably high Deviation: ~0.33mL of fraction #1 was spilled and lost

I precipitated the remaining volume (98µL) of pool#2 (actual conc. 745µg/mL, which matches almost perfectly with recalculated estimate User:Mar/Notebook/2007-6-13) with cold acetone (1+4), centrifuged 16000 xg for 20' @ 4°C, let dry for 20' @ 37°C. The expected yield is 73.01 µg. Reconstituted in same volume (98µL) of 10mM TrisHCl (pH7.5) buffer, shaking @ 37°C for 30'.

= mouse cortical extract (cont) =

Reeler HET: After thawing and spinning, the extract was assayed: 1.884g/mL