Western Blot of PARP-1
Today we will finish the Western Blot using PARP-1 antibody.
Yesterday we blocked your membrane with 10 ml of blocking buffer 5% milk in phosphate buffered saline (1X PBS) with 0.25% Tween (commonly referred to as PBS-T) for 1 hour. We then removed the blocking buffer and added mouse anti-PARP-1 antibody (BD Pharmingen #556362) in 5% milk in phosphate buffered saline with 0.25% Tween (PBST). The anti-PARP-1 was diluted 1:1000 (so you need 10 ul for 10 ml of buffer). These blots were rocked overnight at 4°C. You will begin with the next step. *Note: you will need to save the primary antibody for use by other lab groups.
- Return the primary antibody solution to the conical centrifuge tube and give it back to your instructor (try to recover as much volume as possible). Pour PBST wash buffer onto the nitrocellulose so that it is submerged by approximately 0.5 cm of liquid. Incubate 7 min. on the shaker.
- Pour off the wash buffer into the sink. Add fresh wash buffer and incubate another 7 min. on the shaker.
- Repeat Step 2 once more (a total of 3 7-min. washes).
- Add the HRP-conjugated goat anti-mouse secondary antibody 1:10,000 in 10 ml blocking buffer (i.e. add 1 µl of antibody to 10 ml of blocking buffer). Incubate 45 min. on the shaker.
- Pour off the secondary antibody solution into the sink (you do not need to save this).
- Wash three times with PBST (7 min. each wash) as you did after the primary antibody incubation. After the last wash, remove all residual wash buffer by tilting the blot container onto a paper towel. *Do not proceed with the following steps until your instructor is available to help you with the film exposure and developing. Leave your blot in the final batch of wash buffer until that time. The following directions for performing the ECL may be different depending on which manufacturer’s kit we use. Check with your instructor to see if the following step (ratio of reagents 1 &2) needs to be modified before proceeding.
- Pipet 3 ml of ECL detection reagent 1 into a 15-ml conical tube and add 75 µl of reagent 2. Invert to mix. It is important for the two solutions not to mix until just before you use them! Make sure that you record all the kit information in your lab notebook as the reagents are proprietary.
- Pour the mixed ECL developing solution onto your blot. Make sure all the membrane is in contact with the liquid by rocking the container slightly back and forth by hand for 1 min. Discard the ECL reagents in the sink.
- Working quickly but carefully, use forceps to place your blot onto a piece of paper towel. Using another piece of paper towel, gently blot the nitrocellulose to remove excess liquid.
- Use forceps to place the membrane blot—protein side up—between the two plastic sheets of a page protector that has been taped into an autoradiography cassette.
- With your instructor, take the cassette into the darkroom. Hold the open cassette up to the light for about 20 sec. to activate the glow-in-the-dark StratageneTM logo marker. (This will help in aligning your developed film with the MW markers on the blot.)
- Flip the switch to turn off the normal light and turn on the safe light. (Also, make sure the computer monitor in the room is turned off.) Open the film container and remove one piece of film. Be sure you actually get a piece of film and not one of the thin paper sheets that divide the films. Place the film onto the blot and close the cover of the cassette (be careful not to move the film once it has been placed on the blot). Wait ~2-4 min. then open the cassette and remove the film. Put it in the developer and wait 4 minutes for your film to develop.
Lab 8: Cell Culture
Lab 9: Apoptosis - DNA
Lab 10: Apoptosis - Protein 1
Lab 12: Imaging Presentations