Protocol: Dispense 50 mg of powder to 6 pre-labeled locking 1.5ml tubes
Leave samples at RT ~1 min prior to adding extraction buffer (do one by one better)
When adding extraction buffer vortex for 10sec (full speed)
Extraction buffers used:
1. 20mM HEPES Na pH7.4, 500mM NaCl, 1% Triton X-100
2. 20mM HEPES Na pH7.4, 700mM NaCl, 1% Triton X-100
Keep samples in ice
Vortex again 10sec(full speed)
Extraction buffer (contains protease inhibitor from 100x->1x) @ 1:4 w:v (200 ul of buffer per 50mg of powder)
Sonication:
Program 1, Setting 4 Amp, 5x1 seconds ~ 7J
- 500mM 2x sonicationJ- 7, 7
- 500mM 3x sonicationJ- 8, 6, 8
- 500mM 4x sonicationJ- 7, 8, 7, 8
- 700mM 2x sonicationJ- 6, 7
- 700mM 3x sonicationJ- 8, 8, 6
- 700mM 4x sonicationJ- 8, 7, 8, 9
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Transfer supernatant to new tubes
Measure protein concentration with 660nm assay:
- 500mM 2x sonication- 10.5ug/ml
- 500mM 3x sonication- 10ug/ml
- 500mM 4x sonication- 9.1ug/ml
- 700mM 2x sonication- 10.8ug/ml
- 700mM 3x sonication- 10.9ug/ml
- 700mM 4x sonication- 11.2ug/ml
Sample preparation for Bis-Tris 4-12% gel (before start, preheat thermostat to 70C, at Thermomixer comfort-corner of bench)
Preparation of pellet:
Add 40ul of 1.1x LDS to pellet and heat (72C) for 10minutes to remove proteins from it
Spin down the pellet
Take 2.5 ul from the supernatant (sample name: pellet)
Volume loaded to gel: 10ul
Take the appropriate amount of protein: 25 ug for each sample (lysate, not pellet)
Add in each sample (lysate & pellet): 4xLDS –>1x, DTT (stock concentration is 1M, final concentration should be 50mM DTT, so it’s 1 ul of DTT in every 20 ul of loading volume).
Then heat samples at 70C for 10’ (no shaking)
Spin max spin on top bench centrifuge to reduce bubbles
Remove the comb (gently) out from the cassette by using both thumbs, wash the wells with running buffer and dispose , remove the tape at the bottom of casette, place the cassette
Use 1x MOPS and 350ul ANTIOXIDANT for small gel, double this in big gel.
Loading order:
- Marker
- 500mM 2x sonication- lysate
- 500mM 3x sonication- lysate
- 500mM 4x sonication- lysate
- 700mM 2x sonication- lysate
- 700mM 3x sonication- lysate
- 700mM 4x sonication- lysate
- 500mM 2x sonication- pellet
- 500mM 3x sonication- pellet
- 500mM 4x sonication- pellet
- 700mM 2x sonication- pellet
- 700mM 3x sonication- pellet
- 700mM 4x sonication- pellet
- Marker
Run the gel @200V
When the gel is almost ready, prepare the membrane.
1. Dump MeOH on it, remove it, just to make it wet
2. put in the water for 5 min on the shaker, remove water,
3. put in a transferring buffer for 10 min on a shaker.
Prepare a sandwich BEFORE (transferring buffer in tray) you open the cassette with gel. Keep gel in a transferring buffer until everything for transfering will be ready. Otherwise gel will dry.
Put the transparent/red side down and use 2 filter papers to each side. Gel towards (-) black side and on top of it the membrane (+) transparent/red side
For the big transferring tank - use the ice block. Put the star magnetic bar inside. Put the ice around the tank, place it in a cold room
Wet transfer, 70V for 1.5hr
Transferring buffer reusable
Prapare 5% Milk (2,5 g for 50 ml of TBST buffer)
Blocking- Leave the membrane in milk for 1 hour- or overnight if its at cold-room (4 C)
Wash after milk with TBST (just quick wash)
1st Ab (anti-prkaca) - overnight in cold-room or 1-2 hour in RT. Shaking is required.
Wash with TBST 10 min 3 times.
Change the plastic container (after 2nd wash) because primary antibodies stick to walls- wash the used one with detergent
2nd Ab - 1:10.000 dilution (e.g. for 10ml TBST use 1ul 2nd Ab) - 1 hour in RT.
Wash with TBST 10 min 3 times.
For membrane visualisation
-Spread HRP substrate (2-3ml) evenly around the membrane with pipette (cover whole membrane)
-Before i place it to the visualiser, make sure i drain the HRP substrate by sliding the membrane to the wall of container and wiping the membrane circumferentially with paper- also remove drops from membrane, if needed
Taking pictures of membrane 1 picture every 20-30 seconds-12 pictures total
RESULTS:
