Bio-Rad Sequi-Blot™ PVDF Membrane - Manual (Page 10)

200 µl of 6 N HCl containing 4% thiogly-
colic acid.
2. Seal the tube in vacuo and hydrolyze for
16-24 hours at 110 °C.
3. Extract the liquid containing the amino
acids from the tube, and rinse the PVDF
membrane with an additional 50-100 µl of
6 N HCl.
4. Combine the rinse wash with the extraction
sample and evaporate to dryness.
5. Analyze samples with an amino acid ana-
lyzer.
Note: As a blank control to account for background,
cut a piece of membrane the same size as the
band of interest from a corner of the membrane
and subject it to hydrolysis.
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way to isolate proteins separated in a gel.
Protein samples which have been immobilized
onto PVDF by either spotting, direct adsorp-
tion, or electrophoretic blotting can be
hydrolyzed and subjected to amino acid analy-
sis.
6,7 Amino acid analysis of blotted proteins
using a post-column amino acid analyzer offers
a way to quantitate the sample present prior to
sequencing, as well as a way to determine the
A.A. composition of the proteins of interest.
This data can also be used to determine the effi-
ciencies of the gel electrophoresis and blotting
systems being used for sequencing, whether the
protein of interest is present in enough quantity
to provide useful sequence data, or whether the
quantity of protein is not a problem and amino
terminal blockage might be the cause of
sequencing problems. For amino acid analysis:
1. Place the PVDF membrane piece contain-
ing the sample in a hydrolysis tube and add
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