Note:  Wear gloves- never touch membranes with your bare hands.

Day 1:  Set up protein gels in apparatus as before with small gel plate facing inwards  towards buffer well.  Clamp on either side and fill upper well only with running  buffer.

 Prepare samples by adding SDS loading dye to aliquots (i.e. 6 ul dye plus 24 ul  sample) in an eppendorf tube.  Heat for 5 min and spin down.

 If your gel does not leak too badly, rinse the wells with buffer and fill the lower  well with buffer being careful to just cover the bottom of the gel.

 Load from lanes 1-10 as follows:

 1 10 ul    PCNA control (from us)
 2 24 ul Your crude extract
 3 24 ul Your 600 mM elution
 4 24 ul Your 800 mM elution
 5 24 ul Our E.coli extract (without PCNA from us)
 6 10 ul M.W. Marker
 7 24 ul E.coli extract (without PCNA from us)
 8 24 ul Your 600 or 800 mM elution
 9 24 ul Your crude extract
 10 10 ul PCNA control (from us)
 

 Turn the voltage to 150 and run approximately 30 min until the blue dye in all  lanes has reached the bottom of the gel.

 Turn off the power and carefully remove the gel from between the two plates.   Cut the gel in half down lane 6 (trying to keep some marker on each side).  Place  one half (lanes 6-10) in the Coomassie blue dye to stain.  The other half (lanes 1- 6) will be used for the Western.

 Soak the other half (lanes 1-6) in Western Transfer Buffer (WTB) for 15 minutes.

Western Transfer Apparatus Set-up

 Set up transfer apparatus as follows:
 - Fill a shallow tray with transfer buffer. Place gel holders dark side down in tray.
 - Place a sponge pad down and cover with WTB. Remove air bubbles.
 - Place Whatman paper cut to size over sponge pad.  Remove air bubbles.
Wear gloves and use foreceps to handle the paper.
 - Carefully put the gel onto the moistened filter paper.
 - Cut a piece of nitrocellulose the size of your gel.  Write your name on the top of the nitrocellulose with ball point pen or a VWR marker.
 Be sure to wear gloves and handle the NC membrane GENTLY with foreceps. The membrane is very fragile.
 -  Soak the NC in WTB until completely moistened.
 -  Place the NC on top of the gel being sure to touch the gel only once.  Do this with a minimum amount of buffer to allow for gel/NC contact.
 - Remove air bubbles by rolling a pipet over the membrane and place one piece of 3 mm Whatman paper on top of the NC.  Roll out air bubbles and add more WTB if necessary.
 - Place second sponge pad on top, being sure to remove air bubbles.
 - Compress the sandwich and slide into the box.
 - Surround transfer apparatus in ice.
 - Attach red lead to the front plate and black to the back and transfer
   at 100 mAmps constant current using the BIO RAD power    supply (MAX VOLTAGE is 50 V).
 -  It’ll take 2 hours.
 When the transfer is finished, and with your gloves on, take your gel and nitrocellulose filter apart.  Place your gel in Coomassie Blue stain.  Wrap your nitrocellulose filter in plastic wrap, label, and store at 4C.
 

Note:  You must be prepared and do things right on schedule or this will take all night.

Day 8:  AM:  We will treat your blots with blocking solution for 2 hours.
 Rinse membrane 3 times for 5 min each with TBS and Tween 20.  Discard  washes down the drain.

 Add 15 ml diluted primary antibody (anti-PCNA).  Incubate at least 1 hour  with agitation at RT.

 Pour antibody off into a 15 ml sterile tube and label well.  Give to the TA.

 Wash membrane with TBS and Tween 20 three times, 5 min each time.  Discard  washes down the drain.

 Add secondary antibody by diluting 1ul of the goat anti-rabbit IgG with alkaline  phosphatase conjugate into 25 ml dilution buffer.  Incubate for 1.5 hours  with  agitation at RT.

 Pour the secondary antibody solution down the drain and rinse the membrane 3  times with TBS plus Tween 20.  Allow 5 min per wash.

 Rinse the membrane twice in alkaline phosphatase buffer (at 4C).

 Develop by soaking in solution which contains: 10 ml alkaline phosphatase buffer
  66 ul NBT (50 mg/ml at -20C)
  33 ul BCIP (50 mg/ml at -20C)

 Purple bands should develop to indicate where the PCNA protein is.  Allow the  color to develop a little past the perfect exposure and then rinse with distilled  water.  This color indicator fades with exposure to light, so after the membrane is  dry and you have shown it to everybody, wrap it in foil to preserve it.