Column Buffers: CREB

Bases Buffer is Buffer C (as per Schroter)(as per SRF)pg.27

20mM Hepes pH 7.9

0.1% NP40

0.2mM EDTA

20% glycerol

Lysis Buffer: 0.75M KCl

Need buffers containing 0,0.1M,0.6M & 1.0M KCL in addition to 0.3M,1.5M & 2.0 used in SRF purification pg.27.

Column Buffers

Make 500mls equivalent of base buffer:

10ml 1M Hepes pH 7.9

100ml 10% glycerol

5ml 10% NP40 (or 5.0ml 300mM Octyl glucoside)

200ml 0.5 EDTA

-------

115.2ml /5 = 23ml

Base 1M KCl2.5M KCl dH20

for 0 KCL 46ml 0 0 154ml = 200ml

for 0.1M KCl23ml 10ml - 67ml = 100ml

for 0.6M KCL 23ml 60ml or 24ml 17 or 53ml = 100ml

for 1.0M KCl23ml - 40ml 37ml = 100ml

For final Lysis Buffer add: /10ml prior to use

20ml 1M DTT = 2.0mM

25ml 0.2M PMSF = 0.5mM

10ml 1mg/ml pepstatin = 1mg/ml

10ml leupeptin = 1mg/ml

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

10ml 0.1M Na Orthovalol = 0.1mM

10ml 0.1M Am Molybdase = 0.1mM

500ml NaF @ 1M = 50mM

Add above to 0 0.1M KCl Buffers also prior to use.

Add DTT,PMSF,Na Ortho & Molyb.to rest of buffers prior to use.

Storage Buffer for Column For 500ml

10mM Tris ph 7.5 5ml of 1M

0.3M NaCl150ml 1M or 60ml 2.5M

1mM EDTA 1ml of 0.5M

0.02% NaN₃ 1ml of 10%

CREB Column - Purification Trial #1

Sample #5-C4-1-1 (pg.25)lysed in 4.5ml (3 vol.)0.75M KCl Buffer C (188x10⁶cells)

Thawed on ice.

Pellet 20min.in microfuge at 4℃.

Saved super - but noted pellet not tight and some got into super.

Froze - 80℃.

Thawed sample

Pelleted 20 min.in microfuge - rest of pellet came down.@4.0ml

Transferred to 26ml 0 KCl Buffer C = 1:7.5 dilution = 0.1M KCl

Save @150ml aliquot of undiluted

Added 3mg Herring Testis DNA - 0.1mg/ml (Not necessary.)

When diluted a ppt.formed,resembling stringy DNA.

Pelleted 10min.at 3000rpm on table top at 4 ℃.(Nice pellet formed.)

Saved pellet tried to resuspend in 500ml 0.75M KMg²⁺ very viscous.

Prepared CaRE columns.Pooled column material left over from runs by Morgan - 4.0ml material @ 40mg/ml CaRE/column.

Split into 2 - 2.0ml columns.

Washed with several volumes column storage buffer until settled.

Washed with 5 volumes (10ml each)0.1M KCl Buffer C.

Split cell lysate in 1/2 and ran equal amounts over each column (2X).

Collected flowthru.

Saved 100ml of diluted sample and of flowthru samples.

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

Washed with 10 volumes of 0.1M KCl Buffer C (20ml)each.

Collected 10-2.0ml fractions of washes = pool 1-3,4-10.

All fractions collected were 2.0ml and contained 8mg of insulin as carrier.

Eluted with 4 x 2.0ml (1 vol.)0.3m KCl Buffer C

" " " 0.6M " " .

" " " 1.0M " " .

" " " 1.5M " " .

" " " 2.0M " " .

Sample loaded at 9:30 - first pars by 10:30,second by 11:30.

Washes done by @1:30.

Elutions done by @3:30-4:00

Rinsed column with 2 additional voumes 2.0M.

Rinsed column with 5 volumes storage buffer and sealed off.

100ml aliquots of each fraction were saved separately.

All samples were frozen on dry ice immediately.

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

50ml (25ml)sample

150ml (175ml)dH20

1ml = 5mg insulin

40ml 100% TCA

on ice 1 hours.

Pelleted 15 min.,TCA removed.- Pellets nearly invisible.

Washed with 250ml Ether.

Be sure to vortes wash since TCA needs to "Dissolve" in ether.

At this point a white pellet is ususally seen.

Pellet 15 min.,remove ether,vacuum dry.

Resuspend in 20ml dH20.

Then added 20ml 2X fresh SDS Buffer - made in plastic,BME 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.

0.3M were pretty heavy pellets.

0.6M - smaller pellets.

1.0M - nice pellet in #2 fraction!!!

1.5M 2.0M - almost no pellet.

Might help to increase carrier to 20mg/ppt.

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

Loaded 1/2 sample - 25ml equivalent of cell fractions and 6.25ml equivalent of load,flowthru,pellet fractions (1/4).

Gels: 2-8.5% SDS-Page

23ml 30% acrylamide 3.33ml 30% acrylamide

30ml 1M Tris pH 8.8 2.50ml 1M Tris pH 6.8

26.2ml dH₂O  14.0 dH₂O

0.8ml 10%SDS 0.2ml 10%SDS

460ml 10% APS 100ml 10% APS

50ml TEMED 20ml TEMED