Column
Buffers: CREB
Bases buffer is
Buffer C (as per Schroter) (as per SRF) (pg. 27).
20
mM Hepes pH 7.9
0.1%
NP40
0.2
mM EDTA
20%
glycerol
Lysis buffer:
0.75 M KCl
Need buffers
containing 0, 0.1 M, 0.6 M and 1.0 M KCL in addition to 0.3 M, 1.5 M and 2.0
used in SRF purification (pg. 27).
Column
Buffers
Make
500 ml equivalent of base buffer:
10
ml 1 M Hepes pH 7.9
100
ml 10% glycerol
5
ml 10% NP40 (or 5.0 ml 300 mM octyl glucoside)
200
µl 0.5 EDTA
115.2
ml /5 = 23 ml

Base 
1 M KCl 
2.5 M KCl 
dH_{2}0 

For 0 KCL 
46 ml 
0 
0 
154 ml 
= 200 ml 
For 0.1 M KCl 
23 ml 
10 ml 
 
67 ml 
= 100 ml 
For 0.6 M KCL 
23 ml 
60 ml or 
24 ml 
17 or 53 ml 
= 100 ml 
For 1.0 M KCl 
23 ml 
 
40 ml 
37 ml 
= 100 ml 
For final lysis
buffer add: /10 ml prior to use.
20
µl 1 M DTT = 2.0 mM
25
µl 0.2 M PMSF = 0.5 mM
10
µl 1 mg/ml pepstatin = 1 µg/ml
10
µl leupeptin = 1 µg/ml
100
µl 10 KU/ml trasalol = 100 U/ml
10
µl 0.1 M Na Orthovalol = 0.1 mM
10
µl 0.1 M Am Molybdase = 0.1 mM
500
µl NaF at 1 M = 50 mM
Add above to 0 +
0.1 M KCl buffers also prior to use.
Add DTT, PMSF,
Na Ortho & Molyb. to rest of buffers prior to use.
Storage buffer
for column 
For 500 ml 
10 mM Tris ph 7.5 
5 ml of 1 M 
0.3 M NaCl 
150 ml 1 M or 60 ml 2.5 M 
1 mM EDTA 
1 ml of 0.5 M 
0.02% NaN_{3} 
1 ml of 10% 
CREB
Column  Purification Trial #1
Sample #5C4Ð1Ð1
(pg. 25) lysed in 4.5 ml (3 vol.) 0.75 M KCl + Buffer C (188 x 106 cells)
Thawed on ice
Thawed sample
Prepared CaRE
columns
Flow rate was
consistent and even over entire column run at 1.0 ml 5 min.
Eluted with 4 x
2.0 ml (1 vol) 0.3 m KCl + Buffer C.
" " " 0.6
M " "
" " " 1.0
M " "
" " " 1.5
M " "
" " " 2.0
M " "
Sample loaded at
9:30  first pars by 10:30, second by 11:30.
Rinsed column
with 2 additional volumes 2.0 M.
Rinsed column
with 5 volumes storage buffer and sealed off.
100 µl aliquots
of each fraction were saved separately.
All samples were
frozen on dry ice immediately.
50 µl of each
sample were TCA ppt (25 µl of load; FT + pellet).
On ice 1 + h.
Pelleted 15 min,
TCA removed.
Pellets nearly
invisible.
Washed with 250
µl ether.
Pellet 15 min,
remove ether, vacuum dry.
Resuspend in 20
µl dH_{2}0.
Then added 20 µl
2X fresh SDS buffer  made in plastic, ME added last before use.
Pellet volumes
seemed to correlate with expected fractions.
Wash fractions 
observed gradual decreases in size of pellet as increase in time of wash so by
#9  not much pellet left.
0.3 M were
pretty heavy pellets.
0.6 M  smaller
pellets.
1.0 M  nice
pellet in #2 fraction!!!
1.5 M + 2.0 M 
almost no pellet.
Might help to
increase carrier to 20 µg/ppt.
Samples boiled 3
min and loaded onto 8.5% acrylamide gels.
Loaded 1/2
sample, 25 µl equivalent of cell fractions and 6.25 µl equivalent of load, flow
thru, and pellet fractions (1/4).
Gels: 2Ð8.5%
SDSPage 

23 ml 30% acrylamide 
3.33 ml 30% acrylamide 
30 ml 1M Tris pH 8.8 
2.50 ml 1 M Tris pH 6.8 
26.2 ml dH_{2}0 
14.0 dH_{2}0 
0.8 ml 10% SDS 
0.2 ml 10% SDS 
460 µl 10% APS 
100 µl 10% APS 
50 µl TEMED 
20 µl TEMED 