Purification of 15N Labeled Galectin-1
Protocol
Originally developed by K.P. for non-labeled galectin-1
Modified by H.H., and J.N.
Further modified by S.M. for 15N labeled galectin-1
Date Revised 12/22/11
Chemicals:
Agar
Ampicillin
BL21 Competent Cells
B-PER
DTT
IPTG
b-Lactose
PBS
Plasmid for Gal-1 – pT7IML-1 in expression vector pGEMEX
PMSF
Sodium Azide
Sodium Chloride
Sodium Hydroxide
Tryptone
Yeast Extract
Solutions:
20% Sodium Azide – store at RT
o 20g NaN3
o qs to 100ml dH2O
10% PBS – store at RT
o 80g NaCl
o 2.0g KCl
o 14.4g Na2HPO4
o 2.4g KH2PO4
o Dissolve in 800ml dH2O, adjust to pH 7.4
o qs to 1L dH2O
o Autoclave
Washing Buffer – for affinity column, 0.02% NaN3 in PBS, store at 4°C
o 1ml 20% NaN3 stock solution
o 100ml 10X PBS
o qs to 1L dH2O, adjust to pH 7.5
Equilibrating Buffer – for affinity column, 0.02% NaN3, 8mM DTT in PBS, store at 4°C
o 1ml 20% NaN3 stock solution
o 100ml 10X PBS
o qs to 800ml dH2O, adjust to pH 7.5
o 1.234g DTT
o qs to 1L dH2O
Elution Buffer – for gel filtration, 0.02% NaN3, 8mM DTT, 0.1M b-Lactose in PBS, store at 4°C
o 100ul 20% NaN3 stock solution
o 10ml 10X PBS
o qs to 80ml dH2O
o 123.4mg DTT
o qs to 100ml dH2O
Strip Buffer – for affinity column, 0.02% NaN3, 0.1M NaCl, store at 4°C
o 5.84g NaCl
o 1ml 20% NaN3 stock solution
o qs 1L dH2O
Harvesting Buffer – for cell pellet lysis, use 6-7ml/pellet, 8ml DTT, 1mM PMSF in B-PER, store at RT
o 0.123 DTT
o 0.017g PMSF
o 100ml B-PER
Ampicillin Stock Solution – 25mg/ml, store at -20°C, 1ml/tube
o 1.25g Ampicillin
o qs to 50ml dH2O
o aliquot at 1ml/tube
o use 2 tubes/500ml bottle
IPTG Stock Solution – 100mM IPTG, store at -20°C, 550ul/tube
o 0.238g IPTG
o qs to 10ml dH2O
o mix on rocker at RT for 10-15 minutes to get it into solution, aliquot at 550ul/tube, use 500ul to induce cells
LB Agar/Amp Plates – 100ug/ml Ampicillin
o 250ml LB Broth
o 3.75g Agar
o Autoclave in 500ml screw top bottle at 121°C for 30-40 min. Add 1ml 25mg/ml Ampicillin, swirl bottle to mix. Transfer 20-25ml into 10 sterile 100mm diameter Petri dishes. Allow plates to cool to RT and solidify. Store in cold room (4°C)
SOC Media – 2% tryptone, 0.5% yeast extract, 10mM NaCl, 2.5mM KCl, 10mM MgCl2, 10mM MgSO4
o 2.0g tryptone
o 0.5g yeast extract
o 1ml 1M NaCl
o 250ul 1M KCl
o 1ml 1M MgCl2
o 1ml 1M MgSO4
o qs to 100ml dH2O
o sterile filter and aliquot 90ul/1.5ml tube and store at -80°C
5X M9 Salts Media - (800ml)
o Na2HPO4 · 7H2O 51.2g
o KH2PO4 12.0g
o NaCl 2.0g
o NH4Cl 4.0g
Add 700ml distilled water, pH to 7.4 then bring volume up to 800ml
Autoclave
15N Labeled Working Solution
o autoclaved water 800ml
o 5x M9 salts 200ml
o 20% w/v Glucose (filter sterilize, keep at 4°C) 20.0ml
The following should be syringe filtered and added to working solution:
o *1M MgSO4 2.0ml
o *10mM Thiamine 2.97ml
o *Ampicillin (50mg/ml) 2.0ml
o *100mM CaCl2 1.0ml
Procedure :
Day 1 – Transform Competent Cells with Gal-1 Plasmid
1. Dilute plasmid pT7IML-1 to 1ug/ul
a. 1ul 46ug/ul plasmid stock from -80°C
b. 45 ul PBS
2. In a 1.5ml pre-chilled tube:
a. 20ul competent cells
b. 1-2ul diluted plasmid
Incubate: 2 min on ice
40-60 seconds at 42°C
2 min on ice
Add: 80ul SOC media
Incubate 1 hour at 37°C incubator (200-250rpm)
3. Streak LB Agar/Amp plates with transformed bacteria
a. Turn on Bunsen burner
b. Heat loop and let cool
c. Transfer 15ul transformed bacteria to plate
d. Spread with loop
e. Incubate in 37°C incubator overnight
Day 2 – Grow Transformed Cells
4. Inoculate flasks of 15N labeled working solution
a. Number plates and flasks to match 500ml flasks
b. Transfer 200-250ml 15N labeled working solution to 500ml erlenmyer flasks (one flask per cell plate).
c. Transfer 2 colonies from each plate into one flask using bacteria loop and flaming in between for sterility
d. Incubate at 37°C shaking @ 250 rpm overnight (18-24hr)
Day 3 – Induction of Bacteria
5. Transfer bacteria to IL flask
a. Each 200ml bacteria/500ml flask à 2 x (100ml bacteria in 1L flask)
b. Add the following to each 100ml bacteria in 1L flask
i. 400ml 15N labeled working solution
ii. 1ml 25mg/ml Amp
c. Incubate 1 hour at 37°C, 250 rpm
6. Induce using IPTG
a. Add 500ul 100mM IPTG to each flask
b. Incubate 4 hours at 37°C, 250 rpm
Day 4 – Collection of Galectin-1
7. Centrifuge to collect bacteria
a. Aliquot bacteria solution into 50ml centrifuge tubes
b. Balance and load centrifuge, spin 10 min at 5000 rpm at 4°C
c. Remove supernatant, store pellet at -20°C until use
8. Cell Lysis (Keep on ice)
a. Resuspend each pellet in 2ml Harvesting Buffer
b. Triturate to thoroughly mix
c. Combine cells into 1 bottle
d. Rinse with additional 5-10ml Harvesting Buffer to complete transfer
9. Centrifuge to collect lysate (Keep on ice)
a. Aliquot cell lysis solution into 2ml microfuge tubes
b. Spin in microcentrifuge at 13,500 rpm for 10 minutes, 4°C.
c. Collect supernatant in 50ml conical tubes (contains the 15N labeled galectin-1)
10. Loading Affinity Column (column should be stripped with stripping buffer then equilibrated using equilibrating buffer prior to protein loading), 4°C
a. Slowly load column with lysate being sure not to disturb beads
b. Allow lysate to sit overnight
Day 5 – Affinity Chromatography for 15N labeled Galectin-1 purification
11. Flow-through
a. Open stopcock and adjust flow rate to approx. 1 drop/second
(Unbound protein will flow through column)
b. Add 100ml washing buffer when lysate solution is 1” above resin
c. Allow washing buffer to flow through the column
d. Collect fractions when washing buffer is 2-4” above resin and check absorbance using nanodrop.
i. If absorbance is same as blank, begin elution
ii. If absorbance is higher than blank, add additional 50ml washing buffer
12. Elution – Be sure to collect 500ul elution buffer to use as blank for nanodrop
a. Add 100ml elution buffer to column ONLY after being washed completely in step 11
b. Collect fractions into 2ml microfuge tubes for the duration of the elution. (approx. 45-60min)
c. Read each fraction using nanodrop
d. Pool collected fractions that contain protein, mix, and read absorbance to obtain final protein concentration.
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