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IMMUNOPRECIPITATION PROTOCOL WITH ANTI-CATHEPSIN D ABS
Antibodies were made in rabbits against SDS denatured highly purified recombinant human fibroblast procathepsin D expressed in E. coli. Thus the antigen protein was not glycosylated. A discussion of some aspects of the procedure is found below. SDS IMMUNOPRECIPITATION PROCEDURE Cell Harvest (60mm dish)- depending on the cultured cell line, cathepsin D is visible on a fluorogram in 24 hr, more or less. The labeling media or the chase media (normally 1 mL) is removed from the cells, centrifuged for 5' in microcentrifuge and the supernatant is frozen at -20 deg. The cells are washed three times with CaMgFree-PBS. SDS Cell Lysis Buffer:
heat to 100 deg C. in boiling water bath (for immediate denaturation of proteases) add 0.5 mL per 60 mm culture dish, rock the plate to evenly distribute and lyse cells scrape and then transfer into 1.5 mL centrifuge tube (the lysate should be very viscous, transfer with a wide bore something, like a pasteur pipette) add PMSF to 0.1 mM (0.1 M stock in EtOH) Sonicate until viscosity approaches that of water. For SDS denatured cell extracts, the cells are sonicated with 30-40 pulses with a microprobe Heat Systems Sonciator or until the viscosity is not detectable. for complete denaturation - add 2 uL of 1 M DTT Boil 3-5 min to completely denature and solubilize complexes Add 50 uL of iodoacetamide ( 0.5M IAA) to block all free sulfhydryls (remember this works best at alkaline pH) incubate at room temp for 30 min to 1 h for incomplete denaturation (if the Abs react poorly to completely denatured forms or if the crosslinker precludes reduction of disulfides) add 50 uL of 0.5M IAA and no DTT Add 170 uL of 10% Triton X-100 (this absorbs SDS to prevent denaturation of the Abs), mix well spin in microfuge 5 min full speed (e.g. 12k x g) The media containing secreted labeled proteins were solubilized with by adding SDS to 0.4% and treated as the cell extracts above. remove the extract to clean tube and add Abs Incubate 1-18 h (the more Abs the shorter the time) spin in microfuge 5 min full speed (e.g. 12k x g) remove the supernantant to clean tube add Protein A Sepharose incubate 1-3 h on a rotator spin momentarily in microfuge (only spin long enough to bring down the beads) remove the supernatant (most of it) to a tube (for possible later use) add 1 mL of wash buffer 2.5% Triton X-100
mix with beads by inverting the tubes spin momentarily remove most of the sup and discard (this can be done VERY carefully with an aspirator) repeat the 1 mL addition of wash buffer, spin etc until 5 additions of wash are completed repeat the wash two times with 0.1 M NaCl, 10 mM Tris pH 8.0elute the
beads by boiling for 5min in SDS gel sample buffer (we
use 3% SDS, 20% Glycerol, 80 mM Tris base, 10 mM EDTA, 0.1 M DTT, 0.1%
Bromophenol Blue)
CONSIDERATIONS FOR THE SDS IP: How much antiserum or ABS? If you are going to make quantitative arguments comparing one lane to the next, you must completely precipitate the Ag ie be in Ab excess. The only way to determine this is by titration with different amounts of Ab increasing until no more Ag is found on the gel and by demonstrating that reprecipitation of the extract with another aliquot of Abs does not bring down more Ag. Note that if the IP is incomplete, you may selectively precipitate one species of your protein over another (perhaps another form in complex with other proteins, or modified with a crosslinker etc.) If you are impatient or are having dirty IPs try using alot more abs and decrease the incubation time with the Abs If you overload the gel with Ig you may have problems resolving procathepsin D from single chain cathepsin D. If your antiserum reacts poorly with SDS denatured Ag, you may need more antiserum to obtain complete precipitation. If you cannot use the right amount of Abs without overloading the gel try using a larger gel, fewer cells (if your signal is high enough), or affinity purifying the Abs. To start try 1 - 5 uL of a good antiserum. For cathepsin D IPs, we use about 15 µg of affinity-purified antibodies per 60 mm dish for 1 h at room temp. How much Protein A Sepharose (PAS)? You must use an excess of PAS, ie. the stated binding capacity of the PAS should be several times that of the added Ig. This is hardly ever a problem since the PAS binds 20-30 mg Ig / mL of beads. If you use too little PAS you cannot see what you are doing. If you use to much PAS you cannot efficiently elute the Ag-Ab complex. Do not use more PAS than .5 vol of the maximum volume which can be loaded on the gel. The PAS is swollen in water, washed with water or wash buffer, adjusted to be approximately 50% bead/50% water, mixed and pipetted with a very wide bore pipet tip. Make sure the PAS doesn't settle out during aliquoting to the tubes. With a 5-10 fold excess of PAS 1 h is sufficient at room temp. Cathepsin D - Apparent MW's on SDS gels:
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