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Labeling Proteins with [14C]-Formaldehyde References: J. Cell Biol. (1982) 92:674-686.
 

Summary: 115
Labeling Proteins with [14C]-Formaldehyde
References: J. Cell Biol. (1982) 92:674-686.
J. Biol. Chem. (1979) 254:4359-4365.
Isotope: NEN NEC-039H, 250 ÁCi.
Labeling protein molecular weight standards:
1. Transfer 100 Ál of Biorad MW standard proteins to an Eppendorf tube.
Ě Alternatively, resuspend Pharmacia MW standard proteins in 100 Ál ddH2O.
Ě Perform the following steps at room temperature.
2. Add 1 Ál of 1% SDS (ultrapure) and vortex gently.
3. Add 15 Ál of freshly made 20 mM NaCNBH3 (= 0.1 g in 8 ml ddH2O). Vortex gently.
4. Add 1 Ál of 1 M NaPi, pH 7.5. Vortex gently.
5. Initiate reaction by adding 18 Ál (36 ÁCi) of [14C]formaldehyde (0.25 mCi/125 Ál = 2 ÁCi/Ál).
Vortex gently.
6. Add 15 Ál ddH2O to bring final volume to 150 Ál.
7. Hold at room temperature for 12 - 24 hours.
8. Store at -20░C. The [14C]formaldehyde is completely consumed during the 1 day incubation. There
is no need to quench the labeling reaction or separate free label.
Labeling Concanavalin A:
1. Add 70 Ál of 20 mg/ml Con A to an Eppendorf tube.

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine