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 祃 0.5 EDTA

                           115.2 ml /5 = 23 ml

 

 

Base

1 M KCl

2.5 M KCl

dH20

 

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 祃 1 M DTT = 2.0 mM

                  25 祃 0.2 M PMSF = 0.5 mM

                  10 祃 1 mg/ml pepstatin = 1 礸/ml

                  10 祃 leupeptin = 1 礸/ml

                  100 祃 10 KU/ml trasalol = 100 U/ml

                  10 祃 0.1 M Na Orthovalol = 0.1 mM

                  10 祃 0.1 M Am Molybdase = 0.1 mM

                  500 祃 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% NaN3

1 ml of 10%

 

 

CREB Column - Purification Trial #1

 

Sample #5-C4󕘃 (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 祃 aliquots of each fraction were saved separately.

All samples were frozen on dry ice immediately.

50 祃 of each sample were TCA ppt (25 祃 of load; FT + pellet).

On ice 1 + h.

Pelleted 15 min, TCA removed.

Pellets nearly invisible.

Washed with 250 祃 ether.

Pellet 15 min, remove ether, vacuum dry.

Resuspend in 20 祃 dH20.

Then added 20 祃 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 礸/ppt.

 

Samples boiled 3 min and loaded onto 8.5% acrylamide gels.

Loaded 1/2 sample, 25 祃 equivalent of cell fractions and 6.25 祃 equivalent of load, flow thru, and pellet fractions (1/4).

 

Gels: 28.5% SDS-Page

 

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 dH20

14.0 dH20

0.8 ml 10% SDS

0.2 ml 10% SDS

460 祃 10% APS

100 祃 10% APS

50 祃 TEMED

20 祃 TEMED