Iodination of VWF (and CMP?)

Dominik Haudenschild, 10-28-94 at CBRC

From Loscalzo, J., Fisch, M. an Handin, R.I. "Solution Studies of VWF" , (1985) Biochemistry 24, 4468-4475

 

Outline: Basically a 3 step protocol:

1: Add Iodo-beads to an 125I buffered solution in reaction vial, and incubate at RT.
Suggested time is 5 minutes

2: Add protein in buffer to reaction vial, incubate at RT
Suggested time is 2-15 minutes, neutral pH

3: Separate reaction from Iodo-beads to terminate iodination

Additional 4th step is separation of Iodinated protein from unincorporated 125I
VWF paper uses G-25, 10cm x 0.7cm pre-treated with TBS+5mg/ml BSA
Collect 0.3ml fractions
For analysis ONLY: TCA precipitate, count pellet and supernatant
Achieved 0.1µCi/µg specific activity

 

Detailed Protocol for 94µg of CMP (I would do two or three reactions)

1: Just prior to use, wash beads with 500µl of iodination buffer per bead, and dry beads on filter paper

2: Add 1 bead to a 125µl solution of 1mCi Na125I in iodination buffer in glass tube:
Incubate 5 minutes, (VWF is 15 minutes)

3: Add 1ml CMP @ 94µg/ml
Incubate 2-15 minutes (VWF is 25 minutes with gentle rocking)

4: Remove solution from bead to stop reaction. Wash bead with 125µl of PBS to recover any CMP that may adhere to bead or glass tube

5: Gel filtration to remove excess Na125I or unincorporated 125I2,
-Equilibrate GF-5 column with 25ml Desalting buffer
-Apply 1.25ml sample, allow to permeate gel
-Add 1.25ml desalting buffer, discard flowthrough
-Add 4.75ml desalting buffer, collect labelled CMP
-Column should contain only Na125I or unincorporated 125I2

-Alternative to gel filtration is to dialize against PBS

 

CMP is at 94µg/ml in 150mM NaCl, 15mM Tris pH ~7.3
(Biorad protein assay using BSA as a standard)

Iodination buffer is 150mM NaCl, 15mM Tris pH ~7.3

Desalting buffer is Iodination buffer + 1mg/ml BSA