Biology Reference
In-Depth Information
2. Materials
Unless stated otherwise, all reagents should be of Analar grade.
1.
Sodium borohydride should be dissolved in 0.01
M
sodium hydroxide solution at
5
C immediately prior to use. The dry solid is deliquescent producing an
explosive gas (hydrogen) when wet, consequently, care with storage and han-
dling is essential.
°
2.
The hydrochloric acid used for protein hydrolysis is a constant boiling mixture.
This can be purchased commercially (BDH, Poole, UK) or prepared in the labo-
ratory by distillation of a 50% mixture of hydrochloric acid with distilled water
and collecting the distillate that separates at 110
°
C.
3.
Fibrous cellulose, CF-1, is a commercially available product from Whatman
(Maidstone, Kent, UK).
4.
Filters, for sample preparation, and for both HPLC and amino acid analyzer buffer
filtration, are commercially available (HPLC Technology, Macclesfield, UK).
4mm or 13 mm PVDF syringe filters are used for sample filtration.
5.
A steel 'mortar and pestle'. The 'mortar' consists of a cylindrical block of stain-
less steel (40 mm
40 mm) with a flat-bottomed 10-mm diameter hole drilled
into it to a depth of 30 mm. The “pestle” is also made from stainless steel and
measures 9.5 mm in diameter and 100 mm in length. These dimensions provide a
close sliding fit into the mortar.
×
6.
A source of liquid nitrogen.
7.
An amino acid analyzer equipped with a post-column ninhydrin detection system.
8.
A high performance liquid chromatography (HPLC) system linked to a
fluoresence detector.
9.
Ideally, both of the above should be equipped with computer-based chromatog-
raphy data handling software or a computing integrator.
10.
The reagents and buffers for use with the amino acid analyzer are best purchased
from the equipment supplier. Any alteration to the concentration or pH of such
buffers should be done with great care and any buffers modified in this way
should be passed through a 0.2
m filter prior to use to remove particulate matter.
The buffers should incorporate 0.01% phenol to prevent bacterial spoilage and
should be stored at 15-20
µ
°
C.
11.
All HPLC reagents need to be HPLC grade and 0.2
µ
m filtered prior to use.
12.
A microtiter plate reader fitted with a 570 nm filter and a number of flat-bot-
tomed 96-well microtiter plates are required for the pyrrole assay.
The following reagents are also required for measuring the pyrrole crosslink,
and can all be obtained from Sigma-Aldrich (Poole, UK).
13.
TAPSO Buffer: 2.81 g of 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxy-
propanesulphonic acid (TAPSO) is dissolved in 80 mL of distilled water, adjusted
to pH 8.2 with 1
M
sodium hydroxide and then made up to 100 mL.
14.
TPCK/TAPSO enzyme inactivator: 1.6 mg N-tosyl-L-phenylalanine chloro-
methyl ketone (TPCK) is dissolved in 100
L of ethanol and then taken up to 5 mL
in TAPSO buffer. This solution may be turbid, but the turbidity will be removed
later in the assay by filtration.
µ
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