Biomedical Engineering Reference
In-Depth Information
cally. It is recommended to re-equilibrate the buffer after dilu-
tion and/or mixing with salt- and protein-containing solutions.
Re-equlibrate the buffer after changing the temperature, too.
-
If buffer stock solutions are used, adjust them in a way that the
working dilution has the right pH (pH mostly increases when
buffers are diluted).
-
Some buffer substances interfere with enzymes. They act as
competitive inhibitors, inhibit as products, or withdraw essen-
tial ligands by chelate formation.
-
Since some buffers contain reactive groups as primary amino
groups, they do not suit in covalent coupling of proteins.
-
Monitoring of protein content is often disturbed by UV absorp-
tion of buffers or buffer constituents.
-
Some buffers have excellent characteristics with respect to bi-
ologic compatibility and/or buffering features, but they are so
expensive that large-scale use is nearly impossible.
For an idea of buffering ranges, Fig. 7.6 shows these ranges for some
commonly used substances.
7.4.1 Commonly Used Buffers
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Acetate buffer, pH 4.0, I
0.1
Mix 14.95 ml glacial acetic acid or 261 ml 1 M acetic acid with
7.875 g anhydrous sodium acetate or 96 ml 1 M sodium acetate
and fill up to 1000 ml.
=
-
Barbital acetate buffer, pH 8.6, I
0.05
Dissolve 4.88 g barbital-sodium (Veronal, sodium diethylbar-
biturate) and 3.23 g sodium acetate trihydrate in about 800 ml
ddH
2
O. Adjust pH to 8.6 with 0.1 N HCl (consumption about
30 ml), then fill up to 1000 ml.
=
-
Barbital acetate buffer, pH 8.4, I
0.1
Dissolve 8.14 g barbital-sodium (Veronal, sodium diethylbar-
biturate) and 6.48 g sodium acetate trihydrate in about 800 ml
ddH
2
O. Adjust pH to 8.4 with 0.1 N HCl (consumption about
45 ml), then fill up to 1000 ml.
-
Citrate phosphate buffer, pH 5.0, 0.15M
Dissolve 9.414 g citric acid and 18.155 g Na
2
HPO
4
2H
2
Oin
900 ml ddH
2
O, if necessary, correct pH by addition of diluted
phosphoric acid or sodium hydroxide, then fill up to 1000 ml
with ddH
2
O.
Alternatively, prepare the buffer by mixing 490 ml 0.1 M citric
acid with 510 ml 0.2 M di-sodium hydrogenphosphate.
ยท
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Glycine-HCl buffer, pH 2.5 or 2.8, 0.1M
This is the most suitable buffer for elution in immunoaffinity
chromatography.
Dissolve 7.50 g glycine (aminoacetic acid, glycocoll) in 500 ml
ddH
2
O. Adjust the wanted pH by using 0.1 N hydrochloride
acid, then fill up to 1000 ml.
