Ammonium sulphate purification of
Calreticulin from pancreas
Buffers:
All buffers must be pH before use!
Ammonium sulphate buffer for homogenizing the tissue
2.66 M (NH4)2SO4
0.1 M KH2PO4
pH-7.1
1 mM EDTA
Phosphate buffer for dissolving pellet
0.1 M KH2PO4
1 mM EDTA pH- 7.1
DEAE column buffers
Low buffer:
50 mM NaCl
0.1 M KH2PO4
1 mM EDTA
pH-7.1
High salt buffer:
800 mM NaCl
0.1 M KH2PO4
1 mM EDTA
pH-7.1
Hydroxylapatite chromatography buffers (HTP column)
Column buffer:
10 mM K2HPO4 pH-7.0
Elution buffer:
1000 mM K2HPO4 pH-7.0
Protein storage Buffer: 10mM Tris-HCl
(Low Buffer)
1mM EDTA pH-7.0
Inhibitors:
SL mix 1:2000 dilution
100 mM PMSF – 0.5ml /100ml
200 mM Benzamidine – 0.25ml/100ml
Preparation of SL mix:
Total volume: 100ml in 100% EtOH
Sigma Cat#
Use (mg)
Aprotinin, A-1153
10
Phosphoramidone, R-9382
5
TLCK, T-7254
10
TPCK, T-4376
20
APMSF, A-6664
10
E-64, E-3132
10
Leupeptin, L-2884
5
I Pepstatin, P-4265
2
Aliquot in 2ml fractions and store in -20oC
Take frozen tissue from feezer -800C and thaw on ice O/N
Next day
All work have to be perform in the cold room.
1. Trim away fat and discard, cut up and weight tissue. Use 4ml of ammonium
sulphate buffer for homogenizing tissue per gram of tissue + inhibitors.
2. Homogenize tissue at high speed in blender for one minute, pour
homogenate into centrifuge bottles (balance bottles)
3. Centrifugation:
Beckman centrifuge model #J2-21M
250ml centrifuge bottles (6)
Rotor #JA-14 , 9000rpm , 30 min, 4oC
4. Collect supernatant through gauze and the fast flow filter to separate the
tissue fat .
5. Collect tissue pellet in the homogenizer and re-suspend in one half of the
amount of buffers+ inhibitors used in step 2.
6. Centrifuge homogenate as in step 3.
7. Collect supernatant as in step 4., and combine with previous supernatant
add inhibitors.
Discard pellets in biohazard waste bags.
8. Add 200g of solid (NH4)2SO4 liter of supernatant from step 7. , while
stirring. Stir in the cold room for 30 minutes.
9. pH suspension to 4.7 with phosphoric acid .Stir at least 2 ½ more hours in
the cold room.
10. Centrifuge suspension as in step 3.
11. Pour off supernatant (keep in the cold room) take sample #1 to run on the
gel.
12. Pellet: Use 20ml of phosphate buffer for dissolving pellet + inhibitors per
250ml centrifuge bottle.
13. Pour all suspension in dialysis tubing take samples #2.
14. Dialyze the suspension in 2x 20L of DEAE column low salt buffer, first
change O/N, next change more than 6 hours.
Next day
All work have to be perform in the cold room.
1. Remove samples from dialysis bags
2. Centrifugation:
Beckman centrifuge model #J2-21M
40ml centrifuge bottles (2)
Rotor #JA-17, 9000rpm, 10 min, 4oC
Save supernatant + inhibitors take samples #3
Discard pellet (keep in the cold) take samples #4
DEAE column preparation:
1. Weight ~60 g of dry DEAE beads sock them in plenty of ddH2O
2. Wash beads with ddH2O on the whatman filter then equilibrate beads with
low salt DEAE buffer 5 column volumes (cv), then with 5cv of high salt
DEAE buffer and low salt buffer.
3. Transfer beads into the glass beaker and pour all supernatant mix in the
cold room for 1 hour.
4. Load all mixture onto the column diameter:25mm, length :50cm
5. Collect flow though as one fractions- take samples #5
6. Wash column wit h3cv of low salt buffer collect one fraction - take sample
#6
7. Run 10% SDS -PAGE gel with samples #1-6
Next day:
All work have to be perform in the cold room.
1. Elute bound proteins with linear gradient of 400ml low and high salt buffer,
collect 7ml fractions.
2. Run every second fractions on the 10%% SDS -PAGE gel.
3. Pool together all fractions contained tissue CRT.
4. Pour sample into dialysis bag and dialyze against 2 changes (sample: buffer
1:1000 dilution). First o/n change
Next day:
All work have to be perform in the cold room.
1. Second change at least 6 hours.
2. Remove samples from dialysis bags add inhibitors.
3. Centrifugation:
Beckman centrifuge model #J2-21M
40ml centrifuge bottles (2)
Rotor #JA-17, 9000rpm, 10 min, 4oC
Save supernatant + inhibitors take samples #1
Discard pellet (keep in the cold) take samples #2
HTP column:
Important!
Use new beads each time.
1. Weight 7g capacity of column +70mg (10mg/g dry weight of beads)
2. Column volume 40ml (2cm x 13cm)
3. Weight ~7 g of dry HTP beads sock it in plenty of ddH2O
4. Wash beads with ddH2O on the whatman filter then equilibrate beads with
low salt buffer 5 column volumes (cv), then with 5cv of high salt buffer and
low salt buffer.
5. Transfer beads into the glass beaker and pour all supernatant mix in the
cold room for 1 hour.
6. Load all mixture onto the column
7. Collect flow though as one fractions- take samples #3
8. Wash column with5 cv of low salt buffer HTP column buffer collect one
fraction - take sample #4
9. Store column O/N in the cold room
Next day:
All work have to be perform in the cold room.
1. Elute bound proteins with linear gradient of 400ml low and high salt
HTP buffer, collect 5ml fractions.
2. Run every second fractions on the 10% SDS -PAGE gel.
3. Pool together all fractions contained tissue CRT add inhibitors.
Elution concentration
Use: Amicon Ultra15 Centrifuge Filter Unit from Millipore - cat #UFC9 03024
Concentrate all elution down to very small volume, then exchange high saltBuffer to Protein storage Buffer using the same filter unit .Fill up filter unit with
Protein storage Buffer =inhibitors, spin –repeat exchange at least 3 times.
Remove concentrated protein sample from filter unit collect in the tube (4oC),
combine with other samples from next steps of the purification.
Keep sample all the time @ 4oC.
Sample filter sterilization
At the last step of purification filter sterilize protein sample using Millex Sterile
Syringe Driven Filter Unit-.022m from Millipore, cat# SLGV 033RS.
When all sterilization is done, try to rinse filter unit by filtering just Low-Buffer to
remove the protein reminds from unit.
Protein Assay
Aliquot 200ug samples of tissue CRT freeze in liquid nitrogen and store in -80
freezers.
The final purified sample of tissue CRT runs on the gel 5, 10, and 15 ug
per lane for coomassie blue and sample of 1and 2.5ug per lane for Western blotB
Good Luck