NIA (Nanopro 1000) TRAM PROTOCOL

NIA (NanoPro 1000) TRAM PROTOCOL by Joanna Liliental 1/10/12

CELL LYSIS GUIDELINES:

<100,000 cells Lyse in 10-20l 100,000-1x106 Lyse in 20-50l >1-5x106 cells Lyse in 50-100l 7x106 cells Lyse in 200l

1. Prepare Lysis Buffer (can store up to 1 week at +4C with inhibitors)

1ml CHAPS/Bicine 20l 50x DMSO protease inhibitor (+4oC) 40l25x aquaeous PPase inhibitor (-20oC)

2. Lyse cells: * Maintain pellets on dry ice and transfer to ice when lysing. * Lyse in ___l LB (depending on cell number: see guidelines above) * Pipet up and down and vortex. * Incubate on ice 15 min. * Vortex up and down. * Incubate on ice 15 min. * Spin down 14,000 rpm 10 min (+4oC). * Transfer supernatant to a new vial. * Determine [protein concentration] by BCA. * Use appropriate amount of lysate for NIA.

3. Label tubes: Pt#? 11/16/11, etc.

4. BCA PROTEIN DETERMINATION:  NEED 80l (reagent B/A) per well +2 l lysate (or standard)  Determine how much BCA to make: How many samples + standards (BCA run in duplicates) [#(samples)x80l + 8 (standards)x80l] x2  Make BCA reagent (B/A): (RT bottom left shelf in the hallway) 20 l Reagent A 1 ml Reagent B BSA standards (dilutions made in Lysis Buffer): 0, .25, .5, 1, 2, 4, 8, 10 mg/ml (8 standards)

 Add 2l lysate or standard to a 96well plate in duplicates.  Add 80l BCA reagent (B/A) to each well.  Mix well.  25-30 min (37oC)  Read absorbance at A562nm.  Determine protein concentrations and use calculations to set up sample dilutions for NIA.

5. DETERMINE AMOUNT OF G2 PREMIX TO USE IN THE ASSAY :

(22.5l Premix)x(# of samples) If making 6 samples: 23l x 6 = 140 l Premix needed (make 170l because it is viscous) 12 samples: 276 l Premix needed (make 280l)

6. ADD LADDER to Premix : 3l ladder1 per 100l Premix 5.1l ladder1 per 170l premix 8.1l ladder1 per 270l premix

7. PREPARE 3x PREMIX (G2 pH 3-10)+ladder1 for each lysate : 3x 1x 75% G2 Premix+ladder 22.5l 7.5l 25% Lysate 7.5l 2.5l ------30l 10l

 Need 10l (lysate+Premix) per well, but done in duplicates (x2) = 20l Need to make 30l so it is easier to pipet.

6. PREPARE (DILUTE) Lysates (SAMPLES): (All samples need to be 7.5l per well)  Need 0.1 g/l  If sample=1.1g/l: (1.1g/l)x(Xl) =(0.1g/l)x(30l) X=2.8l sample Add 4.7l Lysis Buffer (Bicine/CHAPS + inh) to make a total of 7.5l  Add 22.5l Premix to all samples in tubes to make a total of 30l.

7.5l diluted sample + 22l Premix (+ladder) = 30 l TOTAL (for each sample)

7. Add Sample+Premix to all wells: Need 10l per well (add from 30l to 2 wells, if done in duplicates).

8. Make antibody dilutions for each Antibody (in AB diluent). 1o AB dilutions (HSP70: 1l in 500l). If the antibody has not been validated: use 1:50 dilution! 2o AB dilutions (1.5l in 150l) Luminol dilutions (1:1 70l rgnt1+70l rgnt2) 2o Biotin (1l in 150l) 3o Streptavidin (1.5l in 150l) 9. Add Antibodies and Luminol at 10 l per well to appropriate rows. 10. Take the plate downstairs (CCSR basement) and follow the NanoPro Operating instructions.

NanoPro1000 Operating Instructions: 1. Open Compass program 2. Instrument  Open Trays 3. Click on RESOURCES. It opens and closes trays. 4. Take out old capillaries box and put new box in (remove the lid). 5. Refill wash, anolyte and catholyte (take bottles out to refill, so there is no spill inside the machine) 6. Click on RESOURCES to close. 7. Dump waste (bottle underneath the NanoPro) to the sink .

8. Refill water bottle with H2O (bottle underneath the NanoPro). 9. Instrument  Autoclean (that takes about 5-10 min). 10. Spin down the plate with samples (CCSR Rm. 0128). 3000 rpm, +4C, 10 min 11. Get plate from CCSR Rm. 0128. 12. Click on SAMPLES to open the tray with plate. 13. Take out any used/old plate. 14. Insert the test plate (leave the lid on) and click SAMPLES to close the tray. 15. File NEW (add new program cycles, add new rows for antibodies, label samples, Abs, etc). 16. VIEW Advanced Protocol 17. Exposures (7 or 8?): 10, 30, 60, 120, 240, 480, 700, 1000sec (signal), 20sec (registration) 18. FileSAVE (save in the correct folder) 19. START RUN!!! 20. HAVE FUN!!!!