Biology Reference
In-Depth Information
0.5 mL 1-o-n-octyl-
-glucopyranoside (Boehringer-Mannheim) to 400 mL dis-
tilled water and mix well until dissolved. Add 0.305 g MgCl 2 · 6H 2 O, 0.166 g
CaCl 2 , 4.38 g NaCl, 5 mL 1 M Tris-HCl, pH 8.0. Make volume up to 500 mL.
Filter sterilize and store at 4
β
°
C.
3.
Plasmin solution: dissolve plasmin (Boehringer-Mannheim) in plasmin digestion
buffer to make a 1 U/mL stock (stable for at least 1 mo in aliquots at -70
°
C).
Dilute to 0.2 U/mL in plasmin digestion buffer immediately prior to use.
4.
5% BSA solution: dissolve 2.5 g bovine serum albumin (BSA) in 50 mL PBS.
Check that pH is neutral, as some BSA preparations are acidic. Filter sterilize and
store in aliquots at -20
°
C (stable for several mo).
5.
Antibodies: anti-LTBP1 antibody is a rabbit polyclonal antiserum against puri-
fied human platelet LTBP1 (Ab39). Characterization and specificity of this anti-
body is described in (2) .
6.
0.5 M EDTA: dissolve 84.06 g Na 2 -EDTA in 400 mL distilled water. Adjust pH
to 8.0 with 1 M sodium hydroxide (NaOH) and adjust final volume to 500 mL
with distilled water.
7.
Radio immunoprecipitation assay (RIPA) buffer: 50 m M Tris-HCl, 150 m M
NaCl, 0.5% sodium deoxycholate (DOC), 1% Nonidet P40. For preparation of
500 mL; dissolve 3.03 g Tris, 4.4 g NaCl in 450 mL dH 2 O, bring to pH 7.3 with HCl.
Add 5 mL nonidet P40, 12.5 mL 20% DOC, slowly bring pH to 7.2 with HCl, bring
volume to 500 mL, filter sterilize and store at 4
°
C (stable for several months).
8.
Immunoprecipitation wash buffer 1 (low salt): 1% Triton X-100, 1% DOC, 0.1%
SDS, 50 m M Tris-HCl, 150 m M NaCl, 10 m M EDTA, pH 7.5. For preparation of
1 L; add 10 mL Triton X-100 to 800 mL dH 2 O, mix well until dissolved. Add
6.06 g Tris, 8.8 g NaCl and when dissolved bring to pH 7.5 with HCl. Add 10 mL
10% SDS (this is commercially available as a 10% stock. Otherwise it can be
prepared dissolving 10 g SDS (Bio-Rad Laboratories, Hercules, CA) in distilled
water to a final volume of 100 mL. Note: care should be taken when weighing out
this reagent, gloves and a face mask should be worn to avoid contact with the
skin and respiratory tract), add 20 mL 0.5 M EDTA. Bring volume to 1 L, filter
through 0.45
µ
M filter and store at 4
°
C (stable for several months).
9.
Immunoprecipitation wash buffer 2 (high salt): 1% Triton X-100, 0.5 M NaCl, 20 m M
Tris-HCl (pH 7.5). For preparation of 1 L; add 10 mL Triton X-100 to 800 mL
dH 2 O, mix well until dissolved. Add 2.42 g Tris, 29.3 g NaCl and when dissolved
bring to pH 7.5 with HCl. Bring up volume to 1 L. Filter through 0.45
µ
M filter
and store at 4
°
C (stable for several months).
10.
3X SDS-PAGE sample buffer (nonreducing): (Electrophoresis quality reagents
are purchased from Bio-Rad Laboratories). Dissolve 2.28 g Tris in 50 mL dis-
tilled water, add 3 g SDS, 1.5 mg bromophenol blue. Adjust pH to 6.8 with HCl.
Make volume up to 70 mL, then add 30 mL glycerol. For 3X reducing sample
buffer add 3 mL 2-
β
-mercaptoethanol. 3X sample buffer should be filtered
through a 0.45-
C (stable for several
mo). To make 1X sample buffer, dilute 3X sample buffer 1:3 with distilled water
(note: SDS will precipitate at cool temperatures, therefore make sure that SDS is
thoroughly dissolved when thawing out frozen aliquots).
µ
m filter and then stored in aliquots at -20
°
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