Cell lysates

General lysis buffers
The choice of lysis buffer depends on what kind of experiment you are doing.

RIPA buffer gives the lowest background, but can denature some kinases. It also has the potential to disrupt protein:protein
interactions.

NP40 buffer (EVF) is less denaturing, but gives a higher background. It is less likely to inhibit kinase activity and disrupt protein
complexes.


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Additives:

EDTA prevents phosphorylation in the lysate. We usually use 2 mmolar.

Sodium vanadate inhibits all tyrosine protein phosphatases. We use 200 micromolar and add it fresh for each experiment from a stock
made in water and stored in a plastic tube at room temperature.

Sodim fluoride is an inhibitor of Serine/Threonine protein phosphatases. We use it at 50 mmolar.

These should be added to both RIPA and NP40/EVF.

Some people like to add Trasylol or aprotinin, a protease inhibitor.

In our experience, there is no need to prepare the vanadate any special way. Storage in glass leads to the appearance of
precipitates.

Phosphate-buffered RIPA is generally the best choice. Phosphate is a good buffer at pH 7.2 and also functions as an inhibitor of
phosphatases. Tris is a poor buffer at pH 7.2 and does not inhibit phosphatases. Tris is helpful if you need to add calcium or
manganese, both of which will precipitate phosphate. Otherwise, phosphate is preferable.

RIPA is: 

1% NP-40 or Triton X-100 
1% sodium deoxycholate 
0.1% SDS 
0.15 molar NaCl
0.01 molar sodium phosphate, pH 7.2 
1% Trasylol, a 1:100 dilution of what comes from Mobay 

If Tris is substituted for phosphate, use 50 mmolar Tris-HCl, pH 7.2 .

Add sodium fluoride and EDTA. The EDTA is very important. The fluoride is less so. Add the vanadate freshly each time you use it.


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NP40 buffer used to be our name for RIPA lacking DOC and SDS. It is now called EVF, which refers to the additives, not the
distinguishing detergent. NP40 buffer can be made with either NP40 or Triton X-100, and can be buffered with either phosphate or Tris,
depending on your needs.

NP40 buffer is:

1% NP-40 or Triton X-100 
0.15 molar NaCl
0.01 molar sodium phosphate, pH 7.2 
1% Trasylol, a 1:100 dilution of what comes from Mobay 

If Tris is substituted for phosphate, use 50 mmolar Tris-HCl, pH 7.2 .

Add sodium fluoride and EDTA. The EDTA is very important. The fluoride is less so. Add the vanadate freshly each time you use it.


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PBS, in the context of immunoprecipitation, is: 

0.15 molar NaCl 
0.01 molar sodium phosphate, pH 7.2 

The PBS used in tissue culture is a more complex physiological phosphate- buffered saline which contains calcium, magnesium, and
potassium
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TN is: 

0.15 molar NaCl 
0.05 molar Tris-HCl, pH 7.2