Kunxin Luo/James Hardwick CNBr Cleavage Procedure
1. Immunoprecipitate the protein and run it on a preparative
gel. CNBr cleavage must be done with protein transferred
to a nitrocellulose filter. Neither Immobilon nor Nylon can substitute.
IMPORTANT: Wash the NC 2X
for 15 min in deionized H2O after the transfer is complete. This
removes any residual Tris-glycine that seems to affect the migration
of the CNBr fragments during SDS-PAGE.
2. Cut out the piece of NC containing the protein and immediately
put it into a microfuge tube containing 200 microliters of 100mg/ml
CNBr in 70% formic acid. CNBr stocks are 200 milligrams/ml in
70% formic acid and are stored at -70°C. 70% formic acid
is made from 98-100% formic acid and MQH2O.
Optional: If you want to know
how many total counts you have in each membrane piece, place
the cut out pieces of filter in H2O and count them (Cerenkov)
before you place them in the CNBr.
3. Incubate the protein in CNBr at room temp. for 1.5hr. Increased
digeston time does not increase yield.
4. Spin 1-5 min in the microfuge. Transfer supe. to a new microfuge
5. Speed Vac dry (30min for 200 microliters).
6. Resuspend in 500 microliters of MQH2O and dry on the speed
vac again (approx 2 hrs.). This should get rid of the residual
Kunxin resuspended in 40 microliters
in order to save time. Resuspending in larger volumes removes
more of the formic acid and results in better resolution of the
7. Count (Cerenkov) both the dried samples and the membrane
pieces they were released from. Your yield should be between
7. Resuspend in 20-40 µl SDS gel sample buffer. The sample
buffer should remain blue. If it turns yellow, residual formic
acid is present and will cause your samples to run badly . Tris
buffer (1 µl of 1M Tris pH 8.0) can be added to raise the
8. Run on a 24% low bis Tricine gel. Use rainbow markers as MW
indicators. It takes approximately 9 hours to run the bromphenol
blue off of a long gel using a constant current of 50 mA. If
you need to resolve multiple bands below 4kD, then use a separating
gel in addition to the stacker and resolving gel. See the reference
9. For reference, a sample with 100 cpm must be exposed for 2-3
days with a screen to see a strong signal.
Anode (lower) Buffer 1 liter
|0.2 M Tris pH 8.9
||24.2 g Tris base
||Bring to 1 liter with water--adjust to pH 8.9
Tricine Cathode (upper) Buffer 1 liter
||12.1 g Tris base
||17.9 g Tricine
||1 g SDS
||MQH2O to 1000ml (don't need to adjust pH)
24% low bis gel--25 ml (one long gel)
||15 ml 40% Acrylamide
||1.36 ml 1% Bis
|1 M Tris
||8.3 ml 3M Tris HCl pH 8.45
||0.25 ml 10% SDS
||20 microliters 100% Temed
||0.20 ml 10% Ammonium persulfate
Stacking Gel--10 ml
||1.33 ml 30% Acrylamide
||1 ml 1% Bis
|1 M Tris-HCl
||3.33 ml 3 M Tris-HCl pH 8.45
||75 microliters 10% SDS
Plus the usual amounts of Temed and Persulfate.
Buffers, pH, stacking gel recipe from:
Schagger, H. and von Jagow, G. (1987). Anal.
Biochem. 166, 368-379.
Acrylamide:Bis ratio for resolving gel from Kunxin's old
Luo, K., and Sefton, B. M. (1990) Transfer of proteins to membranes
facilitates both cyanogen bromide cleavage and two dimensional
proteolytic mapping. Oncogene
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