In Experiments I and II, we saw how genetic analysis can be used to study the functions and properties of genes.  In this experiment, we will introduce you to some of the biochemical techniques that are used to analyze proteins.  The protein of interest here is the yeast proliferating cell nuclear antigen (PCNA) that is encoded by the POL30 gene in S. cerevisiae.  This protein is worth studying because it is a key component of the DNA replication and DNA repair machineries, and it plays a central role in the regulation of cell proliferation.  In the first part of this experiment, we will purify recombinant yeast PCNA that is over-expressed in E. coli. by nickel affinity chromatography.  In part II, we will subject the purified protein to Western analysis and probe the blots with anti-PCNA antibodies.  Keep in mind that it is important to keep good records (volumes, concentrations, O.D.’s etc.) of the various fractions that you will be working with.  These records are necessary to follow the efficiency of the purification protocol.

Day -2:  Streak E. coli strain containing a plasmid bearing either wt or mutant His-tagged  PCNA onto LB-Amp plate  (done for you).

Day -1:  Innoculate 100 ml LB-Amp with colony from plate, and grow overnight with  shaking at 37°C.  (done for you)

Day 1:  10 am- start 1L cultures with 100 ml overnight (done for you).

 1 PM- Pellet cells in 200 ml aliquots with a 6500 RPM, 5 min spin in the GSA    rotor.  Discard supernatant.
 Resuspend each pellet in 25 ml NBB (Native Binding Buffer) and 25 ul of 100    mg/ml stock of egg white lysozyme, transfer to a 50 ml tube and incubate    on ice for 15 min with agitation.
 Sonicate at setting 7 for 4 times with 10 second bursts.  Allow 20 seconds    between bursts and keep on ice throughout.  The sonicator should not touch   any part of the tube.
 Freeze tube in a methanol/dry ice bath and then thaw in a 40°C water bath.  Repeat   2  more times.
 To break up DNA and make the solution less viscous, aspirate through a 20    gauge needle with a syringe.  Repeat 2 more times.
 Pellet cell debris in SS34 rotor at 7000 RPM for 20 min at 4C.
 Freeze cleared lysate in a new 50 ml plastic tube at -70C.  Mark your tube clearly.
 

Day 3   Note:  Do not allow column resin to dry out at any time.

 Remove sample from -70C freezer and thaw.  Mix and reserve 1.5 ml in a well
 marked eppendorf.

 We will use the batch method for isolation of the pure protein.  Remove and  discard supernatant on top of your 3 ml aliquot of Invitrogen ProBond Nickel  column resin.

 Add 5 ml sterile water, invert tube to mix, spin quickly in table top and discard  water.  Repeat 2 times.

 Rinse column material with 5 ml of NBB, as above, repeating one time.

 Add half of your thawed extract to resin, make sure the top is on securely and put  on shaker in cold room for 15 min.  Spin in table top, take off liquid, add  remaining extract, and mix in cold room for another 10 min.

 Take off liquid as before and save in a well marked tube.  Add 5 ml NBB  and  shake for 5 min in the cold room.  Spin and remove liquid, and add it to the  previous sample of unbound proteins (this is now called NBB sample).

 Remove unbound proteins from resin with a 10 ml wash with Native Wash  Buffer (NWB).  Shake for 5 min in the cold room.  Do not discard liquid this  time, collect and label " NWB eluate 1" with  names and date.  Keep on ice.

 Elute bound protein with NWB containing imidazole.  First, use 3 ml of 200 mM  imidazole (5 min. shake).  Collect eluate on ice and label "eluate 2", names and  date.

 Proceed with 3 ml of 400 mM imidazole.  Collect and label eluate.  Then use 3 ml  of 600 mM imidazole and collect and label eluate.  Finally use 3 ml of 800 mM  imidazole and collect and label eluate.  Freeze samples.

Day 8   Thaw samples obtained on Day 3.  Take 24 ul of each eluate and put in well- marked eppendorf.  Add 6 ul of 6X loading dye, mix and boil for 4 min.  Quickly  spin down samples.

 While the samples are boiling, mark the position of the bottom of each well with a  sharpie on the larger plate after carefully sliding the comb (well former) up and out.  Remove tape from the bottom of the gel.  Do not separate the gel plates.  Set  up 10% gels in apparatus with small gel plate facing inwards towards buffer well.   Clamp on either side and fill upper well only with running buffer.

 If your gel does not leak, rinse the wells with buffer and fill the lower well with  buffer being careful to just cover the bottom of the gel.  Load 10 ul of marker lane  1 and then load your samples in the rest of the lanes (crude lysate from normal E.  coli  (provided by us), crude lysate from PCNA -expressing E. coli,  NBB,  NWB eluate  #1, 2, 3, 4, 5, pure PCNA protein).

 Turn the voltage to 150 and run approximately 40 min until the blue dye in all  lanes has reached the bottom of the gel.

 Turn off the power and carefully remove the gel form between the two plates.   Place in Coomassie Blue stain until Wednesday (Day 9).

 Determine protein concentrations of your samples by the BioRad Bradford assay.  Test all fractions (including NBB sample) and compare values to standard curve  of BSA (see handout).  You will likely have to dilute the first several fractions 10  x to get a reading within range of your standards.

Day 9 Destain gel and dry.

Day 10 Go over calculations of purification efficiency.
 
 

Sample Protein Purification calculations  ‘98
 

Sample  Dil. O.D.          Protein Conc.  Vol.   Total Prot.  % PCNA est.
     (mg/ml) (ml)    (mg)  (from gel photo)

Crude X        20x 0.34  1.2  25     30   ?

Unbound 10x 0.28     0.8  22       17   ?

NWB E 1  10x 0.120  0.15    5      1.5   ?

200 mM 20x 0.401  1.6    3      4.8   ?

400 mM 20x 0.261  0.8    3      2.4   <2%

600 mM 10x 0.207  0.3    3      0.9    10%

800 mM 10x 0.100  0.12    3    0.36   30%