Peter Novick Lab, Department of Cell Biology Yale University School of Medicine
http://info.med.yale.edu/cellbio/Novick/Second/Protocols/DSP.pdf
To a concentrated protein sample (I started with a 20 mg/ml lysate), add 1-3 µl of a 20mg/ml DSP
stock (in 100% DMSO).
Incubate for 10-45 min on ice, or one can also incubate at increased temperatures if the complex is
stable. I incubated on ice for 30 min.
To terminate the reaction, add 0.5 volumes of 0.4 M ammonium acetate. Incubate on ice 10 min.
Add SDS to 1% and heat to 65ûC for 10 min. Cool on ice.
Add buffer containing 1% Triton X-100 (immunoprecipitation buffer) to dilute the SDS. To a 50
µl sample I added 700 µl of buffer.
You can next preclear the samples to decrease the resulting background as follows:
Add BSA to 0.05% (BSA in immunoprecipitation buffer)
Add Protein-A Sepharose beads
Incubate on nutator in the coldroom for 1-2 hours.
Spin in coldroom microfuge for 1 min.
Remove supernatant and place in fresh tube
Add serum for immunoprecipitation. The amount added will be variable depending on the
antibody used. For Sec8 precipitation I used 8µg of affinity purified antibody protein (6 µl of aff.
pur. sera) For Sec4 immunoprecipitation, I used 3.5 µl of crude Shy sera.
Incubate on nutator in the coldroom for at least 2 hours. You will have to do a test to determine the
time required to obtain good precipitation. I used overnight incubations in antibody, as a 2 hr
incubation resulted in only 35% the amount of precipitation as a overnight incubation.
Add Protein-A Sepharose beads (60 µl of a 28.1 mg/ml stock in immunoprecipitation buffer) and
incubate 2 hrs on the nutator at 4ûC.
Spin in microfuge for 20-30 seconds. If you are doing a precipitation using a cold (non-
radiolabelled) lysate save some of this first supernatant to do a Western blot to determine the
percent of the total lysate that was precipitated. If the lysate was 35S-labelled, discard the
supernatant in the appropriate waste.
Wash the pellet with at least 4-5 washes of immunoprecipitation buffer. For 2 washes I included
2M urea in the buffer and for another 2 washes I included 500 mM NaCl in the buffer. These
washes helped to reduce the background, especially when using a radiolabelled lysate.
To the final pellet, resuspend in 20-100 µl of 1X sample buffer containing 50 mM DTT (to reduce
the crosslinker and release proteins). Boil for 5 min and load onto gel. For precipitations using a
cold lysate perform Western blot with nitrocellulose transfer.
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