======ICAM-1 D1 Purification ======

    
===== Materials =====
 
//Washing buffer// \\ 
50mM Tris (pH 8.0) \\ 
23% w/v sucrose \\ 
0.5% w/v Triton X-100 \\ 
1mM EDTA \\ 

//Solubilization buffer// \\ 
50mM Tris (pH 8.0) \\ 
6M Guanidine HCl \\ 
1mM DTT\\ 

//Refolding buffer// \\ 
50mM Tris (pH 8.0) \\ 
10% glycerol \\ 
1mM MgCl2 \\ 
3mM Cystamine \\ 
6mM Cysteamine\\  
 
===== Growth and Harvesting of cells=====



1.	Innoculate 250mL of LB Ap with a 5ml starter culture of BL21 DE3 (pET20b/ICAM1 D1)\\ 
2.	Grow for 3-4 hours to an OD600 of approximately 0.5\\ 
3.	Induce with IPTG (1mM final concentration)\\ 
4.	Harvest cells by centrifugation at 3,200 rpm (using benchtop centrifuge) for 10 minutes\\ 
5.	Store cell pellets at -20 C overnight\\ 

===== Cell Lysis and purification of inclusion bodies =====


6.	Resuspend pellet in 10ml of washing buffer and transfer to a Nalgene centrifuge tube (suitable for the Sorvall SS-34 rotor)\\ 
7.	Do three freeze-thaw cycles: freeze in dry-ice and ethanol (~10 minutes), thaw in the 37 C waterbath (~ 10 minutes).\\ 
8.	Fill Nalgene tube up to approximately two-thirds with washing buffer and then sonicate. Use the large tip. Program for 10 s on and 10 s off, for  2 minutes total process time. Use temperature control probe set to 37 C\\ 
9.	Spin down cells in Sorvall SS-34 rotor for 15 minutes at 12,000 rpm\\ 
10.	Remove supernatant and resuspend pellet in 10ml washing buffer\\ 
11.	Fill Nalgene up to approximately two-thirds with washing buffer and then sonicate as before.\\ 
12.	Repeat centrifugation as in step 9.\\ 


===== Solubilization of inclusion bodies =====


13.	Should recover a slightly brown pellet after this centrifugation step – resuspend in ~ 20ml of solubilization buffer using a plastic transfer pipette (try to avoid bubbles).\\ 
14.	Put solubilization mixure at 4C on a stirplate. Stir slowly for ~ 1.5 hours\\ 
15.	Filter solubilization mixture through a 0.45µm syringe.\\ 

===== Protein refolding =====


16.	Add the filtered solubilization mixture to 2L of refolding buffer and allow to mix slowly at 4C overnight. Cover the beaker but do not seal.\\ 
17.	Check for presence of sulfur smell (cysteamine) – it may be necessary to refold for longer if the sulfur smell lingers\\ 
18.	Use a filtertop filter unit to filter the 2L of refolded protein and transfer to the large concentrator\\ 
19.	Use a 5kDa MWCO filter (Millipore) – shiny side facing up. Start concentrator, making sure that solution is flowing slowly. Concentrate until volume is approximately 50ml. It may be necessary to turn of the stirbar as the solution goes below 100ml, as the stirbar may generate bubbles.\\ 
20.	Remove solution from the concentrator and check protein concentration at A280 using the nanodrop (retain a portion of the flow through from the concentration step for a blank). \\ 

===== FPLC =====

21.	Take 500µl of the protein solution and inject into the S75 FPLC column (flow rate should be set at 0.5ml/min. \\ 
22.	Protein should elute at ~17ml.\\ 

===== Protein Characterization =====


23.	Protein may be further concentrated using a centricon, down to one-tenth of the volume. At this point the buffer may also be exchanged\\ 
24.	Measure the protein concentration again using the nanodrop\\ 
25.	Run an aliquot of the protein on SDS-PAGE to verify size and determine purity.\\ 
    
   Edited By Roisin Owens  6/21/07