Biology Reference
In-Depth Information
Ci
35
S-cysteine per mL labeling media immediately prior to use (NEN Life Science
Products, Boston, MA).
as above), 3 m
M
β
-glycerophosphate (use 100X stock as above). Add 100
µ
4.
Lysis buffer: to 500 mL PBS, pH 7.4 add 5 mL Triton-X-100. Filter sterilize and
store at 4
°
C.
5.
50 m
M
ammonium acetate pH 9.0: dissolve 1.93 g ammonium acetate in 500 mL
distilled water. Adjust pH to 9.0 using glacial acetic acid. Filter sterilize and
store at 4
°
C.
2.2. Analysis of Release of LTBP1 and TGF
β
from
35
S-Labeled ECM by Avian Osteoclast Cells
1.
Avian osteoclast culture medium: To 500 mL
-MEM (Gibco) add 1.1 g NaHCO
3
(if medium is not provided with NaHCO
3
), 5% heat-inactivated FCS, 5% heat-
inactivated chicken serum (CS) (serum lots are individually checked from sev-
eral suppliers; heat-inactivate by heating serum to 56
α
C for 30 min), 2 m
M
LG
(use 100X stock as above) and 100 U/mL P/S (use 100X stock as above). Add
6
°
-D-arabino-furanoside fresh to the medium on the day of use
(make a 1 mg/mL stock in PBS, filter sterilize and store for up to 1 wk protected
from light at 4
µ
g/mL cytosine-
β
C). For reduced serum avian osteoclast culture media, reduce
serum to 2.5% FCS and 2.5% CS.
°
2.
Vitamin D
3
solution: 1,25-dihydroxyvitamin D
3
(BioMol Research Labs, Ply-
mouth Meeting, PA) is diluted to give a 10
-3
M
stock in 95% ethanol. This stock
is extremely light sensitive and should be stored at -20
C under argon in a light-
protected vial. The 1,25-dihydroxyvitamin D
3
is added to avian osteoclast cul-
ture media to give 10
-7
to 10
-8
M
. The stock solution should be diluted
immediately before use and this should be done under reduced lighting condi-
tions. This reagent is not very stable in aqueous solution and any unused diluted
solutions should be discarded.
°
3.
Protease inhibitors: (Boehringer-Mannheim Corp, Indianapolis, IN) (a) aprotinin-
make a 10-mg/mL stock in distilled water (stable for 6 mo stored at -20
°
C in
aliquots), add to samples to give a final concentration of 10
g/mL, (b) leupeptin-
make a 2-m
M
stock in distilled water (0.95 mg/mL) (stable for 1 mo stored at
-20
µ
M
, (c)
pepstatin- make a 1 m
M
stock in methanol (0.686 mg/mL) (stable for 1 mo
stored at -20
°
C in aliquots) add to samples to give a final concentration of 2
µ
°
C in aliquots) add to samples to give a final concentration
M
, (d) prefabloc (AEBSF)- make a 100-m
M
stock in distilled water
(23.95 mg/mL) (stable for 1 mo stored at -20
of 1
µ
°
C in aliquots) add to samples to
give a final concentration of 2 m
M
.
2.3. Immunoprecipitation
1.
1
M
Tris-HCl, pH 8.0: dissolve 60.55 g Tris in 400 mL distilled water. Adjust pH
to 8.0 using concentrated HCl. Bring volume to 500 mL.
2.
-glucopyranoside, 3 m
M
MgCl
2
, 3 m
M
CaCl
2
, 150 m
M
NaCl, 10 m
M
Tris-HCl, pH 8.0). For preparation of 500 mL; add
Plasmin digestion buffer: (0.1% 1-o-
n
-octyl-
β
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