The choice of lysis buffer depends on what the cells were labeled with and whether you want to obtain an active kinase. The lowest background is obtained with RIPA buffer without EDTA. EDTA increases the precipitation of both actin and myosin greatly. Since actin is not phosphorylated, this doesn"t matter much for 32P-labeled samples. RIPA is however more denaturing than NP40 buffer and should not be used for fragile enzymes.
32Pi-labeled cells and isolation of protein kinases.
RIPA buffer containing phosphate buffer and supplemented with 100 to 200 µM sodium vanadate, 50 mM sodium fluoride and 2 mM EDTA should be used with cells which have been labeled with 32Pi. It is also the choice for cells from which you want to obtain proteins with protein kinase activity, provided the kinase is stable.
The EDTA should chelate all the Mg++ in the cells and prevent phosphorylation. Phosphorylation can occur following cell lysis, with the cellular pool of ATP serving as the phosphate donor. The vanadate should inhibit most tyrosine protein phosphatases and the fluoride should inhibit some serine/threonine specific protein phosphatases. Whether the additives need to be present in all the washes isn"t known. They may be completely dispensable after the first spin.
Tamara has found that the vanadate works much more effectively if it is added to the lysis buffer freshly!
Phosphate-buffered RIPA is the best choice. Phosphate is a good buffer at pH 7.2 and also functions as an inhibitor of phosphatases.
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, EDTA and/or vanadate as necessary. Add the vanadate freshly each time you use it.
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, EDTA and/or vanadate as necessary. Add the vanadate freshly each time you use it.
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
0.15 molar NaCl
0.05 molar Tris-HCl, pH 7.2
Beware of fragile kinases. The kinase activities of Syk, the p60src proteins of tsRSVs, of p105fps of PRCII virus and apparently of p70fgr of GR-FeSV 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 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.
The purpose of the DTT is to prevent aggregation due to disulfide formation between newly exposed cysteines. Fresh DTT should be present through the initial steps of the immunoprecipitation. Whether it needs to be present after that isn"t known. 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 not to waste lysate, it"s smart to immunoprecipitate in antibody excess. Antibody:antigen equivalence cannot be predicted. 1 µl of Tamara"s anti-p56 antibody will bind all the p56lck in 5 x 105 LSTRA cells. Since LSTRA cells contain 40 fold more p56 than do other T cells, 1 µl should be enough for 107 regular T cells. The amount of antibody 327 necessary to precipitate all of the p60c-src in cells has not been carefully measured.
Bugs: The dogma is that 2 µl of stock bugs will precipitate all of the Ig in 1 µl of rabbit antiserum. Goat sera do not bind to bugs as well as rabbit sera. Therefore use more bugs when you are using a goat antibody. In most cases, the precipitation of rat or mouse sera is only possible if you add some goat anti-rat or anti-mouse serum, usually the same volume as the volume of primary serum that you are using, 15 min prior to the addition of the bugs.
Pellets which contain less than 15 µl of bugs are hard to handle and easy to lose. 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 which has leached out of the bugs while they were being stored, dilute the bugs with the buffer of interest, spin the bugs down and resuspend them in the buffer you want.
Secondary antibodies:
Most rabbit sera bind well to S. Aureus/protein A. So too do most subclasses of mouse monoclonal antibodies. Goat sera bind, 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.
Cell lysis.
I"m a firm believer in lysing the cells in the cold room! 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. 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. 60 min at 17K in a 1.5 ml eppendorf tube in the old adaptors in the SM24 rotor is generally more than enough. 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.
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.
Packing down the DNA goo.
Many people like to add 30 µl of washed Staph. A. bugs 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.
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 remove the goo with the pipet and throw it away. Generally its all in one gooey strand 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. With large volumes of lysate, the lysate can just be shot in. For small volumes it is necessary to put the lysate directly into the drop of serum. Obviously this necessitates the changing of eppendorf tips.
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.
