Biology Reference
In-Depth Information
2.1 pH Buffers
We use one of the following pH buffers as a component of the
blocking solution. Buffer components are sequentially dissolved in
90 ml water, adjusted to pH 7.4 with HCl or NaOH, and fi nally
made up to 100 ml with water.
1. Tris-HCl 50 mM: 0.605 g Tris (tris(hydroxymethyl) amino-
methane) base or 0.758 g Trizma pre-set crystals (Sigma,
T7693) (
see
Note 2
).
2. TBS (Tris-buffered saline): 0.121 g Tris base (fi nal concentra-
tion 10 mM) or 0.152 g Trizma pre-set crystals, 0.877 g NaCl
(150 mM).
3. PBS (phosphate-buffered saline): 0.818 g NaCl (140 mM),
0.022 g KCl (2.95 mM), 0.032 g KH
2
PO
4
(2.38 mM), and
0.108 g Na
2
HPO
4
(76.1 mM).
2.2 Blocking Buffer
The blocking solution is made of buffer to provide the necessary
pH for antibody binding and of various agents which help to
reduce unspecifi c staining (
see
Note 3
).
Use a magnetic stirrer at moderate speed (
see
Note 4
) to mix:
10 ml Tris-HCl, TBS or PBS (
see
Note 5
), 1 % BSA (100 mg;
see
Note 6
), 0.1 % Tween 20 (polyoxyethylene-sorbitan monolaurate;
10 mg;
see
Note 7
), and 50 mM glycine (37.6 mg; for aldehyde
fi xed cells only;
see
Note 8
).
2.3 Antibody
Solutions
Antibodies are diluted in blocking buffer. The working solutions
should be prepared immediately before use and centrifuged (maxi-
mum speed of microfuge or up to 10,000 ×
g
for 5 min) in order to
remove larger aggregates.
Stock solutions of most primary antibodies should be stored at
≤
−20 °C and frozen in aliquots in order to prevent repeated freez-
ing and thawing; some primary antibodies require storage at 4 °C
or at room temperature. For each primary antibody the optimum
concentration must be tested (
see
Note 9
). Stock solutions of
secondary antibodies are usually stored at 4 °C and often can be
used for months and even years. For secondary antibodies start with
dilutions given in the manufacturer's data sheets (
see
Note 10
).
2.4 Miscellaneous
1. Equipment: magnetic stirrer, pH meter, microfuge, shaker,
and pipettes with adjustable volumes between 0.1 and 10
μ
l,
l.
2. Petri dish with a diameter of 15 cm or larger. Alternatively,
several small Petri dishes can be used.
3. Wet chamber (plastic box with rack to support Petri dish or
plastic box lined with wet fi lter paper).
4. Parafi lm.
5. Fine, antimagnetic tweezers (e.g., Dumont no 7;
see
Note 11
).
6. Disposable Pasteur pipettes.
7. Lint-free fi lter paper.
10 and 50
μ
l, and 100 and 1,000
μ
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