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	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 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% NaN3	1 ml of 10%

CREB Column - Purification Trial #1

Sample #5-C4–1–1 (pg. 25) lysed in 4.5 ml (3 vol.) 0.75 M KCl + Buffer C (188 x 106 cells)

Thawed on ice

• Pellet 20 min in microfuge at 4°C.
• Saved super - but noted pellet not tight and some got into super.
• Froze –80°C.

Thawed sample

• Pelleted 20 min in microfuge - rest of pellet came down at 4.0 ml.
• Transferred to 26 ml 0 KCl + Buffer C = 1:7.5 dilution = 0.1 M KCl.
• Save at 150 µl aliquot of undiluted.
• Added 3 µg Herring Testis DNA - 0.1 µg/ml (not necessary).
• When diluted a ppt formed, resembling stringy DNA.
• Pelleted 10 min at 3000 rpm on tabletop at 4°C (nice pellet formed).
• Saved pellet and tried to resuspend in 500 µl 0.75 M KCl - very viscous.

Prepared CaRE columns

• Pooled column material left over from runs by Morgan - 4.0 ml material at 40 µg/ml CaRE/column.
• Split into two 2.0 ml columns.
• Washed with several volumes column storage buffer until settled.
• Washed with 5 volumes (10 ml each) 0.1 M KCl + Buffer C.
• Split cell lysate in half and ran equal amounts over each column (2X).
• Collected flow thru.
• Saved 100 µl of diluted sample and of flow thru samples.

Flow rate was consistent and even over entire column run at 1.0 ml 5 min.

• Washed with 10 volumes of 0.1 M KCl + Buffer C (20 ml) each.
• Collected 10–2.0 ml fractions of washes = pool 1–3, 4–10.
• All fractions collected were 2.0 ml and contained 8 µg of insulin as carrier.

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.

• Washes done by at 1:30
• Elutions done by 3:30–4:00

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).

• 50 µl (25 µl) sample
• 150 µl (175 µl) dH₂0
• 1 µl = 5 µg insulin
• 40 µl 100% TCA

On ice 1 + h.

Pelleted 15 min, TCA removed.

Pellets nearly invisible.

Washed with 250 µl ether.

• Be sure to vortes wash since TCA needs to "dissolve" in ether.
• At this point a white pellet is usually seen.

Pellet 15 min, remove ether, vacuum dry.

Resuspend in 20 µl dH₂0.

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: 2–8.5% 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 µl 10% APS	100 µl 10% APS
50 µl TEMED	20 µl TEMED