Chapter6: Random Errors In Chemical Analysis
Section: Chapter Questions
Problem 6.16QAP
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Question
Convert the amount of spiperone in each sample to the concentration in the 1 ml total volume of assay tube.
I have calculated amount of 3H spiperone in pmol in each dilution in second picture. To converted concentration in 1 ml total volume of each assay tube do I need to consider dilution factor？
![ABCDELI
A.
C.
D.
E.
F.
G.
H.
NB. The concentration given above will be the TOTAL spiperone concentration, not necessarily
the concentration of 'H-spiperone. The latter needs to be calculated using the SPECIFIC ACTIVITY
of the radioligand and the MAX data (see later).
Preparation of cell membrane
The membrane preparation was diluted in HEPES IA buffer to a concentration of 250μg/ml, so
that 100μl of the diluted membrane solution can be placed in each assay tube to give 25µg protein.
20nM solution
10nM solution
5nM solution
2.5nM solution
1.25nM solution
0.6nM solution
0.3nM solution
0.15nM solution
Preparation of assay tubes
24 assay tubes were required to measure non-specific binding, and 24 were used to measure
total binding of ³H-spiperone to membrane fragments. Each of the 8 spiperone concentrations
were assayed in triplicate; 8 concentrations in triplicate requires 24 tubes, made up as follows:
700μl HEPES IA buffer added to 48 assay tubes.
●
●
.
100μl of 30μM (+)-butaclamol added to the Non-Specific Binding tubes.
100μl of 30μM (-)-butaclamol added to the Total Binding tubes.
100μl of each [H]-spiperone concentration added to the appropriate assay tubes.
●
100μl of the membrane preparation (25µg) added to each of the assay tubes.
Tubes (1.0 ml in total volume) were incubated for I hour in a 25°C waterbath.
Harvesting of assay mixtures
Membrane preparations were harvested on filter paper under vacuum. The filters, which will have
'trapped' membrane fragments and any ³H-spiperone bound to them, were assayed for '³H using
a scintillation counter.
100μl of each ³H-spiperone solution (A-H) were also counted - this formed the MAX data, and
represents how much ³H-spiperone was added to each of the tubes. (NB. Remember that this is
the same amount of H-spiperone in each dilution that was added to the assay tubes, and therefore
will
in th
FUL
Think
is yor](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2F94617a6a-025f-466f-89a1-9321a86532ed%2F1e812007-8aa8-48d7-9c15-3c0d33fec7ef%2Ffye6jdl_processed.jpeg&w=3840&q=75)
Transcribed Image Text:ABCDELI
A.
C.
D.
E.
F.
G.
H.
NB. The concentration given above will be the TOTAL spiperone concentration, not necessarily
the concentration of 'H-spiperone. The latter needs to be calculated using the SPECIFIC ACTIVITY
of the radioligand and the MAX data (see later).
Preparation of cell membrane
The membrane preparation was diluted in HEPES IA buffer to a concentration of 250μg/ml, so
that 100μl of the diluted membrane solution can be placed in each assay tube to give 25µg protein.
20nM solution
10nM solution
5nM solution
2.5nM solution
1.25nM solution
0.6nM solution
0.3nM solution
0.15nM solution
Preparation of assay tubes
24 assay tubes were required to measure non-specific binding, and 24 were used to measure
total binding of ³H-spiperone to membrane fragments. Each of the 8 spiperone concentrations
were assayed in triplicate; 8 concentrations in triplicate requires 24 tubes, made up as follows:
700μl HEPES IA buffer added to 48 assay tubes.
●
●
.
100μl of 30μM (+)-butaclamol added to the Non-Specific Binding tubes.
100μl of 30μM (-)-butaclamol added to the Total Binding tubes.
100μl of each [H]-spiperone concentration added to the appropriate assay tubes.
●
100μl of the membrane preparation (25µg) added to each of the assay tubes.
Tubes (1.0 ml in total volume) were incubated for I hour in a 25°C waterbath.
Harvesting of assay mixtures
Membrane preparations were harvested on filter paper under vacuum. The filters, which will have
'trapped' membrane fragments and any ³H-spiperone bound to them, were assayed for '³H using
a scintillation counter.
100μl of each ³H-spiperone solution (A-H) were also counted - this formed the MAX data, and
represents how much ³H-spiperone was added to each of the tubes. (NB. Remember that this is
the same amount of H-spiperone in each dilution that was added to the assay tubes, and therefore
will
in th
FUL
Think
is yor
![Table 4 Amount of [H]-spiperone in each assay tube
Sample Amount of [³H]-
spiperone (pmol)
A
B
C
D
E
F
G
H
1.35
0.69
0.36
0.19
0.09
0.05
0.02
0.01](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2F94617a6a-025f-466f-89a1-9321a86532ed%2F1e812007-8aa8-48d7-9c15-3c0d33fec7ef%2Fzzy5en_processed.jpeg&w=3840&q=75)
Transcribed Image Text:Table 4 Amount of [H]-spiperone in each assay tube
Sample Amount of [³H]-
spiperone (pmol)
A
B
C
D
E
F
G
H
1.35
0.69
0.36
0.19
0.09
0.05
0.02
0.01
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