Biomedical Engineering Reference
In-Depth Information
µ
should contain 10 - 200
g of protein, and fill up with ddH
2
Oto
µ
l each. Pre-incubate at 30
◦
C for 3 min, and then start phos-
phorylation by addition of 20
200
µ
l of Soln. C.
Stop the phosphorylation: either by 50-fold dilution with ice-
cold Soln. J and centrifugation at 0
◦
C with 50 000 to 100 000
gfor
30 min (in the case of membrane phosphorylation), or by addition
of 1 ml of ice-cold Soln. K after 10 s to 5 min.
For determination of total
32
P-phosphorus incorporation, filter
the precipitate over a glass fiber filter and count for radioactivity.
If electrophoresis is intended, collect the precipitate by centrifu-
gation with 5000
×
×
g and dissolve the pellet in the appropriate
sample buffer. Choose the electrophoresis system with respect to
the different pH stability of phosphorylation products.
6.4 Iodination with [
125
I]-Iodine Reagents
Caution!
125-Iodinemay be liberatedduring iodination evenwhen
solutions areused.Work ina special hood! Check your thyroidgland
after 6 and 24 h if you have worked with iodination reagents.
Iodine becomes incorporated into proteins either oxidatively, or
enzymatically, or electrochemically. Oxidative incorporation uses
organochemical oxidants, such as, for example, chloramine T
or Iodo-gen (1,3,4,6-tetrachloro-3
α
α
-diphenyl glycouril). Enzy-
matic incorporation is done by means of lactoperoxidase. By these
methods iodine is introduced into phenyl (tyrosyl) residues of the
protein.
Conjugation of a protein with the radiolabeled Bolton-Hunter
reagent in the first place modifies lysine side chains.
,6
6.4.1 Chloramine-T Protocol
/
125
I solution, 3.7 GBq
AN
ml
Solutions/Reagents
B . 5M sodium phosphate buffer, pH 7.5
C 0mM sodium phosphate buffer, pH 7.5
D5mg
/
ml chloramine-T (sodium N-chloro-p-toluenesulfonam-
ide) in Soln. C
0.3% 2-mercaptoethanol (v/v) in Soln. C
5
E
F
0.1% serumalbumin (w/v) or 0.1% gelatin (w/v) or 2% heat-
inactivated serum (v/v) in Soln. C
G
0.2% NaI (w/v) in Soln. F
Prepare Soln. D, E, and G immediately before starting the experi-
ment.
/
5
To avoid reduction of disulfide bonds, use 0.2 mg
ml tyrosin instead of
2-mercaptoethanol.
