Tissue Harvest for Protein Gels
Clear arteries well in situ, and after removal rinse and flush with Ringer¡¦s in a petri dish. Blot tissue to dry, and snap freeze in liquid N2. Tissue can be frozen at -70C indefinitely.
1. Fill cooling chamber with liquid N2, and place metal pestle in chamber. Fill pestle with N2 and cool ceramic mortar by immersing in pestle. Let N2 stop boiling before adding tissue. Put tissue in and grind slowly with mortar until tissue is pulverized, and powder feels smooth. Remove pestle from cooling chamber and let N2 evaporate, scraping powder down sides of pestle as evapouration occurs. DO NOT LET TISSUE THAW DURING THIS PROCEDURE!!!!
2. Pick up powder with pre-cooled sterile spatula, and put directly in lysis buffer. Mix well. Use 100 ul lysis buffer for each rat carotid and 200 ul for each rat thoracic aorta.
3. Centrifuge sample in Eppendorf centrifuge at 13000 rpm for 3-4 minutes at r.t.
4. Transfer supernatant to a fresh tube, measuring amount you are taking off.
5. Remove 10 ul of supernatant and put in a fresh tube for protein assay.
6. Add Sample Buffer 1:1 to remaining supernatant, and freeze all tubes at -70C.
Cell Lysis Buffer (General Purpose)
1 ml 10% SDS
100 ul 100mM PMSF(stock in 100% EtOH)
10 ul 10 mg/ml leupeptin
Make up to 10 ml with 50 mM Tris (pH7.6)
2X Sample Buffer
10 ml 0.5M Tris (pH 6.8)
10 ml 10% SDS
10 ml glycerol
1 ml 0.1% bromphenol blue
Make up to 50 ml with ddH2O.
For reducing samples add ƒÒ-mercaptoethanol to a final concentration of 5%.
Store sample buffer at 4C.
Standard curve is 0-1 mg/ml BSA in 100 ug/ml increments. Use Pierce BCA kit.
1. Dilute lysis buffer 1:5 with ddH2O- 0.5 ml lysis buffer to 2.5 ml ddH2O.
2. Dilute BSA stock to 1 mg/ml with diluted lysis buffer - total volume required 1 ml.
3. Add 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 ul of 1 mg/ml BSA solution to labelled eppendorf tubes. Add (in reverse order) 0,10,20,30,40,50,60,70,80,90 ul of dilute lysis buffer to tubes. Mix.
4. Dilute tissue samples 1:5 with ddH2O: take 5 ul and add to 25 ul ddH2O.
5. Prepare reagent: 50 parts A to one of B. If you need 10 ml, add 200 ul B to 10 ml A.
6. Add 10 ul of standard or sample to each well on plate. Run standards and sample in duplicate.
7. Add 200 ul of reagent mix to each well.
8. Incubate plate at 37C for 30 min. Read the plate on reader with shaking, at absorbance 562 nm.
9. Determine concentration of samples by interpolation from the standard curve.
1. Once gel apparatus is assembled, insert comb to proper position and mark edge of separating gel 1 mm below comb edge.
2. Separating Gel
4.6 ml ddH2O
2.5ml 1.5M Tris (pH8.8)
2.7 ml 40% acrylamide
100 ul 10% SDS
100 ul 10% APS
6 ul TEMED
For a zymogram gel, replace 1 ml of ddH2O with 1 ml of 1% gelatin solution, or 1 ml of 1% ƒÑ-casein solution.
3. Use a pasteur pipette to slowly pour gel between glass plates up to mark on glass plates. Layer with ddH2O, and rock back and forth to ensure that interface is even. Leave 15-20 min to polymerize.
4. Stacking Gel
3.4 ml ddH2O
0.63 ml 0.5M Tris (pH6.8)
0.83 ml 40% acrylamide
50 ul 10% SDS
50 ul 10% APS
5 ul TEMED
5. Pour off water from top of gel. Use a pasteur pipette to load stacking gel on top of separating gel. Carefully insert combs between plates, making sure there are no bubbles on comb. Top of well heads on the comb should be submerged. Leave 30 min to polymerize.
6. After the gel has polymerized, snap gels into the gel apparatus, one on either side. It helps to prewet the gaskets with running buffer, this makes the chamber more leakproof.
7. 5X Running Buffer
Trizma base 9g
SDS 3g or 30 ml of 10% SDS
dH2O to 600 ml
Store at 4C.
Dilute the stock 5X runn