Bart's Cookbook--

General Principles of Immunoprecipitation

RIPA buffer
NP40 buffer
PBS
TN
Boiling in SDS
Antisera
Bugs
Secondary Antibodies
Cell Lysis
Pre-clearing
Tamara' s abbreviated protocol


Lysis buffer.

The choice of lysis buffer depends on what kind of IP 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.


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.


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.



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


TN is:

0.15 molar NaCl
0.05 molar Tris-HCl, pH 7.2

Beware of fragile kinases. The kinase activities of Syk and the p60src proteins of tsRSVs, are labile. Most of their activity is lost in RIPA. For these proteins, it is important to use NP-40 buffer, containing EDTA and phosphatase inhibitors, for both the lysis of cells and the washing of IPs. It is also ABSOLUTELY essential to work only in the cold room with these proteins!


Boiling in SDS. For samples that are to be totally denatured, lyse the cells in a small volume--0.1 ml per 35 mm dish--of 0.5% SDS, 1 mmolar fresh DTT, and some buffer, maybe 0.05 molar Tris-HCl, pH 8.0. Scrape them up, boil for 30 to 60 sec and then dilute them with 4 volumes of RIPA without SDS but containing 1 mmolar DTT. People sometimes use a RIPA correction buffer.

The purpose of the DTT is to prevent aggregation due to disulfide formation between newly exposed cysteines. Fresh DTT should be present throughout the immunoprecipitation.

The reason to use fresh DTT is that it is possible that the DTT will lose its reducing power if stored at a dilute concentration. It's simplest therefore to make up a fresh batch of RIPA each time you need it from RIPA without DTT and a 1 molar stock of DTT.



Antisera. So as maximize signal, it's important to immunoprecipitate in antibody excess. Antibody:antigen equivalence cannot be predicted. It obviously depends on the abundance of the protein and the titer of the antiserum. Operationally, every serum should be tested in a titration experiment to determine the amount of lysate that saturates 1 or 2 microliters of an antiserum. Use of more lysate that the antiserum can bind does not increase signal, it only increases background. Additionally, all that you are measuring when you do an IP this way is the the amount of antigen that your antibody can bind.

Bugs: The dogma is that 2 µl of stock bugs--Pansorbin--will precipitate all of the Ig in 1 µl of rabbit antiserum. So in theory you don't need a large volume of bugs to bring down your IP, but..

Pellets that contain less than 15 µl of bugs are hard to handle and easy to lose. The yield is often 0%. Therefore, irrespective of the amount of antiserum used, it is a good idea to use at least 15 µl of bugs. Surprisingly, excess bugs do not seem to increase the background.

The bugs ought to be in the same buffer as the cell lysate. To accomplish this, and to get rid of anything that has leached out of the bugs while they were being stored, dilute the stock bugs with the buffer of interest, spin the bugs down and resuspend them in the buffer you want.


Secondary antibodies:

Rabbit sera bind well to S. Aureus/protein A. So too do most, but not all, subclasses of mouse monoclonal antibodies. Mouse IgG2a is best, 2b next, 3 next and IgG1 is bad. Goat sera bind (despite what people say), but not as well as rabbit sera. Rat antibodies bind poorly to Protein A. If you are using an antibody that binds poorly to protein A, you should use a second antibody, usually from a rabbit or a goat, to facilitate binding of the antigen/antibody complexes to the bugs. The second antibody will bind to the bugs.


Cell lysis.


I'm a firm believer in lysing the cells in the cold room! As soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin to occur. These occur at MUCH higher rates at room temperature than they do on ice. Therefore, the medium should be aspirated in the coldroom, the cells should be washed with cold Tris or PBS, cold lysis buffer should be added and the cells should be scraped with a cold rubber policeman. 20 min is a traditional interval for cell lysis. Whether 20 min is really necessary to solubilize the protein that you're interested in isn't known. It may be unnecessarily long. Surprisingly, backgrounds seem to be somewhat lower if the cell lysate in kept concentrated, 0.25 to 0.30 ml per 35 mm dish. Pelleted suspension cells should be resuspended gently in the lysis buffer. Adherent cells should be scraped off the dish but then allowed to dissolved for 20 min on the dish. Scrape them to the side to remove the lysate.

The lysate should be clarified by centrifugation at 4°C for 60 min at 15K in a 1.5 ml eppendorf tube in the refrigerated TOMY centrifuge in the lab. The microfuges in the cold room are not satisfactory because they heat up to room temperature in 15 minutes. For samples to be assayed for kinase activity, 30 min is probably long enough, since background is lower in these experiments.

For boiled lysates, a spin of 90 min is essential unless you add bugs as described below. Otherwise the DNA gooes everything up.

Even if you're not using 0.5% SDS, nuclear lysis can be a serious problem, especially with mammalian cells. Be gentle. During lysis, all I do to the cells is scrape them off the dish with a rubber policeman, let them sit, scrape them again and transfer to the centrifuge tube. Inclusion of EDTA in the lysis buffer helps. So does the use of NP40 buffer.

Packing down the DNA goo.

Many people like to add 30 µl of washed Staph. A. bugs--Pansorbin--to the lysate prior to centrifugation to help pack down any released, gooey DNA. This can cut the clarification spin time in half and has no obvious deleterious effect.


Pre-Clearing.


When the background in an immunoprecipitation is high, some people like to pre-clear the lysate by adding either normal serum and bugs, or bugs alone, to the lysate and then spinning them out. The theory is that this will drag down anything that could get trapped in an IP. This can in fact help reduce the background in some circumstances. Theoretically, it should always help. In my experience however, it often makes no difference at all.



If the nuclei/DNA in the pellet insist on getting into your eppendorf tip after the spin, the best solution, besides re-spinning the sample, is to keep it stuck in the pipet tip, lift it out, and throw it away. Generally its all in one gooey clump, like a miniprep, and the remaining liquid will yield low background IPs.

It is easier to add the lysate to the antibody than vice versa. Therefore I usually set up all the tubes for the experiment with antibody in them before the clarifying spin is finished.

Because antibodies are relatively stable, you don't need to do this in the coldroom. However, lysates should be added to the tubes with antibody in it in the cold room With large volumes of lysate, the lysate can just be shot in. If done carefully, this can be done without touching the tip to the tube and therefore without switching tips. For small volumes it is necessary to put the lysate directly into the drop of serum. Obviously this necessitates the changing of eppendorf tips for each tube.

Immunoprecipitates which are allowed to form overnight seem to have a higher background than ones worked up right away. This may be due to time dependent aggregation or denaturation of cellular proteins. 45 min of incubation of antibody and lysate and 20 min with bugs seems long enough.


The best way to store IPs is as dryish pellets--after aspirating the buffer--at -20°C. They are more stable like this than in sample buffer. You can store them as pellets for several days at -20°C. Samples in sample buffer deteriorate after a week at -20°C. They are very stable at -80°C.

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