23 mocr purification 9 oct

Western blotting of fplc fractions : 80 minutes transfer at 15 V. Blocking started at 2:30 pm for 15 minutes.

Got no bands in Western. Primary 1:2000 anti His (mouse), Secondary 1:10000 goat anti mouse HRP.  

Digesting the 23CFMocr plasmid with BamHI and Eco RI (after and before the Ssp1 site on MOCR) to see if a 700 bp band (204x3 + 100bp from vector) is released. Yes there is a band of 700bp.

The band was given for mass spec and was not 23 CF. The insert had a ecoRI site at the start which created a frame shift. cloned again with new primers. 

Ran SDS gel of FPLC fractions  prestained ladder, 5, 6, 7, 8, .......17

Lysis buffer :  Hepes pH 7.2, 150mM NaCl, 5% Glycerol, 10mM BME, 0.1% Triton X100, 5mM Imidazole.
Sonication for 30 min + 6min + 15 min at 20% amplitude on SBL5 sonicator with the broad tip.
Spin down at 18 k on JA 20 rotor for 30 min at 4 degrees.
Binding to nickel beads (ml) for 1 hour in cold room (5:35 pm to 6:35 pm)
Injected 8 ml of Elution (5E1+3E2) into S75 column.
Ran 12% SDS gel - prestained ladder, lysate(10ul), BAW(40ul), Eln1(40ul), Eln2(40ul), BAE(40ul) 9:30pm

    prest.lad         lysate          BAW                     Elution 1               Elution2                   BAE