Biomedical Engineering Reference
In-Depth Information
Single-stranded nucleic acids are easily and with high efficiency
transferred to membranes for hybridization experiments.
References
Locker J (1980) Anal Biochem 98:358
Rickwood D, Hames BD (eds.) (1984) Gel electrophoresis of nucleic acids:
a practical approach. IRL Press, Oxford
Voytas D (2000) Current protocols in molecular biology. 2.5A. Wiley, New
Yo r k
2.2.3 Identification of Phosphoamino Acids
(Paper Electrophoresis)
Amino acid paper electrophoresis has sufficient sensitivity and
reproducibility for amino acid analysis. If analysis by HPLC is not
available in or around your lab, paper electrophoresis may be an
alternative.
A
glacial acetic acid/pyridine/ddH
2
O 50:5:945 (v/v/v), pH 3.4
Solutions/Reagents
B
ninhydrin spray: 0.3 g ninhydrin dissolved in 100 ml n-butanol
and supplemented with 3-ml glacial acetic acid
6 N
hydrochloric acid
Electrophoresis is done on ready-to-use cellulose plates or on
chromatography paper (e.g., Whatman No. 3MM, Schleicher and
Schuell No. 2040b, Macherey and Nagel MN) of 20 cm length.
The protein or peptide sample is hydrolyzed in a closed tube
at 110
◦
C for 2 h
16
. After hydrolysis the samples are lyophilized,
dissolved in a small volume of ddH
2
O and lyophilized again.
The start area on the plate (strip) is marked with a pencil at the
cathodicside.Ifseveralsamplesshouldbeanalyzedinthesamerun,
the start areas should have a distance of 1.5 - 2 cm. A plastic sheet
covers the starting zone and the plate is sprayed with Soln. A until
it is humid but not wet.
The absolutely dry, lyophilized samples are dissolved in ddH
2
O
to a concentration of 0.2 - 1 mg
/
/µ
ml (1.5 - 5 pmoles
l) per amino
µ
acid. Up to 5
l of the samples are spotted on the marked start.
The connection between electrode chambers of a horizontal
electrophoresis apparatus filled with buffer A and the cellulose sup-
port is made by wetted paper bridges. To avoid liquid moving, the
buffer level must be the same in both chambers. A glass plate, laying
on the paper bridges, covers the separation plate cooled to 0 - 4
◦
C.
Important!
Take care for effective and constant cooling during the
electrophoresis.
16
The hydrolysis conditions of phosphoamino acids with respect of sta-
bility and reproducibility of quantitative determination are discussed
by Bylund and Huang (1976). Anal Biochem 73:477).
