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Isolation of Protein from Tissue

  1. Place the tissues on labeled aluminum foil and immediately place in dry ice. It is imperative that the tissues stay cold so that protease do not have time to act on the protein.
  2. Place the tissues in a round bottom tube and add Brij buffer with protease inhibitors added. Add about 1ml Brij/tissues that equals about 100l in volume. Brij with inhibitors on ice before use.
    Brij 150 (Lysis buffer)
    Tris 1M 1ml
    EDTA 0.5M 0.4ml
    NaCl 5M 3ml
    Brij 96 10% 8.75ml
    NP40 10% 1.25ml
    QS to 100ml with H2O.
    Protease inhibitors (add to the amount that you will need). Keep the solution on ice after addition of inhibitors.
    Leupeptin 1:1000
    Aprotinin 1:1000
    AEBSF 1:1000
    (For 10ml use 10l of each inhibitor.)
  3. Use the blender to disperse tissues into the buffer. use for about 5 seconds and then place the samples on ice again to keep it from getting warm. Wash the blender between samples with PBS.
  4. When samples are in solution (or as close as you can get them), transfer to 1.5ml eppendorf tubes and centrifuge in the cold room for 10 minutes at full speed. Remove the supernatant which contains the protein and place in a new eppendorf on ice.
  5. Do a protein assay using elisa reader.
  6. Store remainder of sample at -20C in a non-frost-free freezer. Freezing and thawing of protein samples degrades them. Label samples with date or experiment number and protein concentration, if known.
  7. Thaw samples on ice. Remove 50l of protein, in a new eppendorf tube. Freeze remainder of samples. Bring samples up to 25l with Brij buffer and equal amount of 2X reducing buffer. Also make a tube of markers by using 10l of rainbow marker, 15l of lysis buffer, and 25l of reducing buffer. Boil samples for 5 minutes using a tube holder which keeps the lids from popping off. Centrifuge briefly to collect sample at bottom of tube. It is now not crucial to keep the samples cold. They are stable after addition of reducing buffer. I usually store them on ice anyway until ready to load on the gel, though.

Preparation of Protein Lysates from Lymphoblasts or Fibroblasts

Collect cells (lymphoblast or fibroblast) from tissue culture flask and wash 3 times with 1X phosphate buffer saline (PBS), pH 7.4. I add 100l Brij buffer (for cells collected from 75mm flask). Sonicate the cell pellets for 45 seconds and keep the pellet on ice for 2 minutes. Repeat sonication two more times. keep the pellet immediate after sonication on ice. here onwards pellet should be on ice, otherwise protein will be degraded, and quickly so. After three sonications, spin the lysate at 14000 RPM for 10 minutes in the cold room using a microcentrifuge. Now the protein lysate is ready to ship. If you are shipping overseas please use enough dry ice in your shipping box.

If you want to run a western, assay the protein using elisa method or any other method. For longer storage, please store protein lysates in -80C. For an immediate western, take equal volumes of protein lysates and 2X reducing buffer (loading dye), boil the lysates with reducing solution for 5 minutes and then cool it down. Spin again the protein lysate for 5 minutes at 14000 RPM in the cold room and then load on a gel. Always use rainbow marker or any other protein marker to estimate the protein sizes.

Reagents

Protein lysate buffer (Brij buffer):

Tris 1M 1ml
EDTA 0.5M 0.4ml
NaCl 5M 3ml
Brij 96 10% 8.75ml
NP40 10% 1.25ml

Make up to 100ml with ddH2O.

Protease inhibitors:

Leupeptin 25M
Aprotinin 25M
AEBSF 25M

For 100ml Brij buffer add 100l of at least two protease inhibitors.

Reducing solution:

Bromophenol blue pinch
0.5M Tris pH 6.8 2.5ml
Glycerol 2.0ml
10% SDS 2.0ml
ddH2O 2.5ml
Beta-mercaptoethanol 1ml
TOTAL 10ml

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This document was created for the neurogenetics laboratory run by P. H. Reddy, Ph.D. in the NSI of OHSU. Page last updated: 18 Sept 2001.