Purification of 15N Labeled Galectin-1

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.

Subculturing Cells Protocol

Subculturing Cells Protocol

Author:  Sterling Mikkelson

Date Revised: 12/14/11

Cell Line(s):  B16F10, 2H11, FSaII, MDCKII, A549, HUVEC**, Fibroblast, HepG2*

Complete Media (Sterile Filtered): 

RPMI 1640 – 15% Fetal Bovine Serum (FBS) + 1% Penicillin/Streptomycin

**HUVEC – 20% FBS+ 1% Penicillin/Streptomycin

                        *EMEM – 10% FBS – 1% Penicillin/Streptomycin

Materials:

·         Pipette Controller

·         Confluent (80%) cell flask

·         1 mL aspirating pipette

·         Hank’s Balanced Salt Solution (HBSS)

·         5 mL pipette

·         0.25% Trypsin

·         0.05% Trypsin (HUVEC)

·         Complete Media

·         10 mL pipette

·         15 mL conical tube

·         Centrifuge (1500 rpm)

·         P1000 Pipettor

Procedure:

1.    Hold T-150 flask upright and using a 1mL aspirating pipette connected to vacuum line, aspirate media from flask

2.    Add 10 mL HBSS to rinse cells by gently rocking in a circular motion

3.    Remove HBSS by aspiration

4.    Add 5 mL of Trysin/EDTA and incubate at 37°C for 5-10min or until 90% of cells have become detached.

5.    Remove flask from incubator and add 6 mL of appropriate media to the cells containing trypsin.

6.    Collect cell solution with 10 mL pipette, and transfer to 15 mL conical tube.

7.    Cap and centrifuge for 5 minutes at 1500 rpm.

8.    Aspirate supernatant and add 3-5 mL fresh media.  Use gentle pipetting to break cell pellet.

9.    Count cells under a hemacytometer. (http://www.microbehunter.com/2010/06/27/the-hemocytometer-counting-chamber/)

10.  Add approximately 5x10⁵ cells from cell stock into T-150 flasks and add ≥15 mL complete media.

11.  Incubate flask at 37°C

EC Migration Assay

 2H11 Migration Assay Protocol

Mayo Lab (Dec. 2011)

Sterling Mikkelson

Materials:

·         Ibidi 24-well culture inserts (http://www.ibidi.de/products/disposables/E_80XXX_CI_StemCell_family.html)

·         Coating Matrix (Gelatin 0.2%, Collagen 0.01%, Fibronectin 1.0%)

·         RPMI 1640 (10% FBS, 1% Pen-Strep)

·         0.25% Trypsin

·         Hemacytometer

Procedure:

Ø  Day 1

·         Coat each well with desired matrix and incubate for 1 hr. prior to preparing cells for plating

·         Aspirate media from stock 2H11 flask

·         Add 5 ml 0.25% Trypsin and incubate for 5 min.

·         Add 6 ml RPMI media to flask containing trypsin, pipette up and down 3-5 times, and add to 15 ml centrifuge tube.

·         Spin at 1500 rpm for 5 min.

·         Aspirate supernatant and add 3-6 ml media (depending on pellet size)

·         Count cells using hemacytometer (ideal amount of cells should be between 15-50 cells per square)

·         Determine cell concentration (Will need 30,000 cells/ 70 ml)

·         Remove coated culture wells from incubator and aspirate coating.

·         Plate calculated cell concentration (30,000 cell/ 70 ml) into each well and incubate 18-24 hours

Ø  Day 2

·         Prepare compound of interest at desired concentration in a total volume of 400 ml

·         Remove 24-well plate from incubator and carefully remove media in each well using a 200 ml pipettor

·         Using a tweezer, grasp the top corner of the insert and pull up and towards you to remove

·         Slowly, add solution (400 ml total volume) to each well, making sure wells are evenly coated

·         Use inverted microscope at Biomedical Image Processing Lab (BIPL)

·         Under 4X objective magnification, capture migration activity at several time point (0 - 24hr)

Peptide Purification by RP-HPLC

**Note: This protocol is written with the instrument settings already saved.  Anginex was the designed peptide of interest used for this protocol.**

Peptide Sample Prep and RP-HPLC

Protocol (12/08/11)

By: Sterling Mikkelson

Instrument Used:  Beckman Coulter System Gold 166 Detector

Peptide Sample Preparation:  *note: concentration may vary depending on peptide being purified*

·         Remove peptide powder from -20°C Freezer

·         Weigh out 150mg and add to 50 mL Distilled Water for a final concentration of 3mg/mL

·         Sonicate w/ heat for 15 minutes followed by vortexing until powder is completely dissolved

·         Label 50 mL conical tube peptide solution “Crude Anginex – 3mg/mL” and store at 4°C

HPLC Startup:

·         Be sure solvent lines are placed in appropriately labeled bottles

o   (A = Water + 0.1% TFA;  B = Acetonitrile + 0.1% TFA)

·         Double click 32 Karat Software icon on desktop

·         Double click UV icon

·         Turn on UV lamp by clicking on lamp image (right center of screen)

·         Lamp will switch from blinking yellow to pink when lamp is warmed up

·         Open HPLC startup (File > Methods > Open > startup.met)

·         Click on blue arrow located on the top right

·         In box next to Sample ID, type in t

·         Data Path: D:\public

·         In box next to Data File, type in t

·         Click Start (run will take approx. 19 min.)

HPLC Peptide Sample Run:

·         Open HPLC run (File > Methods > Open > peptide25b.met)

·         Click on blue arrow located on the top right

·         In box next to Sample ID, type in peptidename_date_number of run

·         Data Path: D:\Data

·         In box next to Data File, type in peptidename_date_number of run

·         Click Start

·         Change Flow Rate to 6.00mg/mL by clicking on Flow Rate box (will vary depending on peptide). Click OK

·         Rotate injection knob counterclockwise to the load position.

·         Load syringe with 4.8 mL (max) peptide solution

·         When box in lower right reads “waiting for trigger,” inject peptide solution in injector port

·         With syringe still in the injector port, rotate knob clockwise until it stops then remove syringe

·         Collect samples at each peak over the duration of the run for mass-spec. analysis

·         Print a copy of the run (right click on graph > utilities > print) and label peaks where samples were taken

HPLC Shutdown:

·         Switch solvent lines to bottles labeled 70% Acetonitrile

·         Open HPLC shutdown (File > Methods > Open > shutdown.met)

·         Click on blue arrow located on the top right

·         In box next to Sample ID, type in t

·         Data Path: D:\public

·         In box next to Data File, type in t

·         Click Start (run will take approx. 19 min.)

·         Lamp will automatically shut off

·         Close program by clicking on “X” located at the top right corner of screen