Editing Prepare-Cel1-from-Celery (section)

== Protocol 1  ==

Note:<br>â¢ Have ready all PMSF and Tris-Buffer solutions<br>â¢ Thaw aliquots of PMSF solution immediately before use<br>â¢ Use a sieve to remove floating debris<br><br>

1.&nbsp; Prepare celery for juicing:<br>&nbsp;&nbsp;&nbsp; a. Cut off the bottom from each bunch to separate stalks<br>&nbsp;&nbsp;&nbsp; b. Trim off tops, leaves, and tough, white bottoms from each stalk<br>&nbsp;&nbsp;&nbsp; c. Rinse with water and dry with paper towels<br>2.&nbsp; Blender celery at 4° C to obtain 1.5 L juice<br>3.&nbsp; Make a 0.1 M Tris-HCL, pH 7.7, 100 ÎŒM PMSF solution in celery juice<br>&nbsp;&nbsp;&nbsp;&nbsp; a. per L juice add: 50 ml 2M Tris pH 7.7<br>&nbsp;&nbsp;&nbsp;&nbsp; b. per L juice add: 1 ml 0.1 M PMSF<br>4.&nbsp; Centrifuge the juice solution at 15,000 g for 20 minutes, 4 °C<br>5.&nbsp; Decant and save supernatant, discard pellet<br>&nbsp;&nbsp;&nbsp;&nbsp; a. Pellet: fibrous and sticky, bright green<br>&nbsp;&nbsp;&nbsp;&nbsp; b. Supernatant: cloudy greenish-yellow<br>6.&nbsp; Bring supernatant to 25% ammonium sulfate<br>&nbsp;&nbsp;&nbsp;&nbsp; a. per L supernatant add: 144 g (NH4)2SO4<br>&nbsp;&nbsp;&nbsp;&nbsp; b. Volume will increase marginally (3%)<br>7.&nbsp; Mix solution gently for 1 hour, 4 °C<br>8.&nbsp; Centrifuge solution at 15,000 g for 45 minutes, 4 °C<br>9.&nbsp; Decant and save supernatant, discard pellet<br>&nbsp;&nbsp;&nbsp;&nbsp; a. Pellet: small and sticky, green or light gray<br>&nbsp;&nbsp;&nbsp;&nbsp; b. Supernatant: cloudy brownish-yellow<br>10.&nbsp; Bring supernatant to 80% ammonium sulfate<br>&nbsp;&nbsp;&nbsp;&nbsp; a. Per L supternatant add: 390 g (NH4)2SO4<br>&nbsp;&nbsp;&nbsp;&nbsp; b. Volume will increase significantly (25%)<br>11.&nbsp; Mix solution gently for 1 hour or overnight, 4 °C<br>12.&nbsp; Centrifuge solution at 15,000 g for 90 minutes, 4 °C<br>13.&nbsp; Save pellet, decant and discard supernatant<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; a. Pellet: large compared to previous pellets, yellowish-brown or beige<br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; b. Supernatant: transparent brownish-yellow<br>14.&nbsp; Store protein-precipitate pellet at 4 °C (weeks) or -80 °C (indefinite) 

=== Dialysis<br>  ===

'''Caution:'''<br>â¢ Use Gloves in All Steps, Note that PMSF is TOXIC.<br>â¢ PMSF will recrystallize at 4 °C â thaw and use immediately<br>â¢ PMSF will degrade over time in aqueous solutions â do not allow to sit in solution<br>

'''Prepare 5 L Buffer B''':&nbsp; <br> 

{| cellspacing="1" cellpadding="1" border="1" style="width: 374px; height: 146px;"
|-
| a. 0.1 M Tris pH 7.7<br>
| add 50 ml 2 M stock
|-
| b. 0.5 M KCl
| add 250 ml 2 M stock
|-
| c. 0.01% Triton X-100 
| add 5 ml 20% stock
|-
| d. 100 ÎŒM PMSF
| add 5 ml 0.1 M stock
|}

'''Resuspend pellets in a volume of Buffer B equivalent to 1/10th the initial volume of celery juice'''<br>&nbsp;&nbsp; a. For a 1.5 L prep, resuspend in a 150 ml solution of protein in Buffer B<br>&nbsp;&nbsp; b. Scrape pellet from wall of centrifuge bottle using a metal spatula<br>&nbsp;&nbsp; c. Smear or break down large chunks of pellets for easier solvation<br>&nbsp;&nbsp; d. Vortex gently by hand and avoid over-foaming solution<br>&nbsp;&nbsp; e. Transfer buffer and pellets from tube to tube until all pellets are combined<br>&nbsp;&nbsp; f. Resuspended pellet should be homogeneous and free of debris<br> 

'''Add suspended protein to dialysis tubes'''<br>

'''Dialyze in 5 or 6 changes of 5 L Buffer B'''<br>&nbsp;&nbsp; a. Stir solution slowly to avoid drawing dialysis tubes down into the stir bar<br>&nbsp;&nbsp; b. Allow last dialysis to proceed for at least 4 hours or overnight<br>

'''Collect the dialyzed protein solution and aliquot'''<br>&nbsp;&nbsp; Aliquot into labeled and dated 1.5 or 2.0 ml tubes<br>

'''Store aliquots at -80 °C'''<br>