Lysis buffers
Lysis buffers differ in their ability to solubilize proteins, with those containing sodium dodecyl sulfate (SDS) and other ionic detergents considered to be the harshest and therefore most likely to give the highest yield.
The main consideration when choosing a lysis buffer is whether the chosen antibody will recognize denatured samples. When this is not the case, it will be noted on the antibody datasheet, and buffers without detergent or with relatively mild non-ionic detergents (NP-40, Triton X-100) should be used.
Protein location and lysis buffer choice
Protein location Buffer recommend
Whole cell NP-40
Cytoplasmic Tris-HCl
Tris-Triton
Membrane NP-40 or RIPA
Nuclear RIPA or use nuclear fraction protocol*
Mitochondria RIPA or use mitochondrial fraction protocol*
Lysis buffer recipes:
NP-40 buffer
150 mM sodium chloride
1.0% NP-40 (Triton X-100 can be substituted for NP-40)
50 mM Tris pH 8.0
This is a popular buffer for studying proteins that are cytoplasmic or membrane-bound, or for whole cell extracts. If there is concern that the protein of interest is not being completely extracted from insoluble material or aggregates, RIPA buffer may be more suitable as it contains ionic detergents that will more readily bring the proteins into solution.
RIPA buffer (radioimmunoprecipitation assay buffer)
150 mM sodium chloride
1.0% NP-40 or Triton X-100
0.5% sodium deoxycholate*
0.1% SDS (sodium dodecyl sulfate)
50 mM Tris, pH 8.0
*Can be prepared as a 10% stock solution, which must be protected from light.
RIPA buffer is useful for whole cell extracts and membrane-bound proteins, and may be preferable to NP-40 or Triton X-100-only buffers for extracting nuclear proteins. It will disrupt protein-protein interactions and may therefore be problematic for immunoprecipitations and pull-down assays.
In cases where it is important to preserve protein-protein interactions or to minimize denaturation, a buffer without ionic detergents (eg SDS) and ideally without non-ionic detergents (eg Triton X-100) should be used.
Cell lysis with detergent-free buffer is achieved by mechanical shearing, often with a Dounce homogenizer or by passing cells through a syringe tip. In these cases, a simple Tris buffer will suffice, but as noted above, buffers with detergents are required to release membrane- or cytoskeleton-bound proteins.
Tris-HCl buffer
20 mM Tris-HCl, pH 7.5
Tris-Triton buffer (cytoskeletal proteins)
10 mM Tris, pH 7.4
100 mM NaCl
1 mM EDTA
1 mM EGTA
1% Triton X-100
10% glycerol
0.1% SDS
0.5% deoxycholate
All four of these buffers will keep at 4°C for several weeks or for up to a year if divided into aliquots and stored at -20°C.
Protease and phosphatase inhibitors
As soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin. These events can be slowed down significantly if samples are kept on ice or at 4°C at all times and appropriate inhibitors are added fresh to the lysis buffer.
Ready-to-use cocktails of inhibitors from various suppliers are available but you can make your own cocktail.
Inhibitor
Aprotinin
Trypsin, chymotrypsin, plasmin
2 µg/mL
Dilute in water, 10 mg/mL. Do not re-use thawed aliquots.
Leupeptin
Lysosomal
5–10 µg/mL
Dilute in water. Do not re-use thawed aliquots.
Pepstatin A
Aspartic proteases
1 µg/mL
Dilute in methanol, 1 mM.
PMSF
Serine, cysteine proteases
1 mM
Dilute in ethanol. You can re-use the same aliquot.
EDTA
Metalloproteases that require Mg2+ and Mn2+
5 mM
Dilute in dH20, 0.5 M. Adjust pH to 8.0.
EGTA
Metalloproteases that require Ca2+
1 mM
Dilute in dH20, 0.5 M. Adjust pH to 8.0
Sodium fluoride
Serine/threonine phosphatases
5–10 mM
Dilute in water. Do not re-use once defrosted.
Sodium
orthovanadate
Tyrosine phosphatases
1 mM
Dilute in water. Do not re-use once defrosted.
Sodium orthovanadate preparation
Perform all steps in a fume hood.
Prepare a 100 mM solution in double distilled water.
Set pH to 9.0 with HCl.
Boil until colorless. Minimize volume change due to evaporation by covering loosely.
Cool to room temperature.
Set pH to 9.0 again.
Boil until colorless.
Repeat this cycle until the solution remains at pH 9.0 after boiling and cooling.
Bring up to the initial volume with water.
Store in aliquots at -20°C. Discard if samples turn yellow.
Preparation of lysate from cell culture
Place the cell culture dish on ice and wash the cells with ice-cold PBS.
Aspirate the PBS, then add ice-cold lysis buffer (1 mL per 107cells/100 mm dish/150 cm2 flask; 0.5 mL per 5x106 cells/60 mm dish/75 cm2 flask).
Scrape adherent cells off the dish using a cold plastic cell scraper, then gently transfer the cell suspension into a pre-cooled microcentrifuge tube. Alternatively cells can be trypsinized and washed with PBS prior to resuspension in lysis buffer in a microcentrifuge tube.
Maintain constant agitation for 30 min at 4°C.
Centrifuge in a microcentrifuge at 4°C. You may have to vary the centrifugation force and time depending on the cell type; a guideline is 20 min at 12,000 rpm but this must be determined for your experiment (e.g. leukocytes need a very light centrifugation).
Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant and place in a fresh tube kept on ice, and discard the pellet.
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