User:Mar/Notebook/2007-6-15

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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:

#  init.vol. init.conc. conc'd vol.  conc.factor
#1 1.6878g - 6.139mg/mL -  46 mg      37x
#2 2.8736g - 6.153mg/mL -  89 mg      32x
#3 3.0735g - 5.833mg/mL - 220 mg      14x
#4 2.8639g - 6.070mg/mL -  76 mg      38x
#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 [1], shaking @ 37°C for 30'.

mouse cortical extract (cont)

Reeler HET: After thawing and spinning, the extract was assayed:

1.884g/mL