Does the calculated curve go near the data points?
If the curve doesn't go near the data, then something went wrong with the curve fit, and the "best-fit" values of Bmax and Kd should be ignored.
Were sufficient concentrations of radioligand used?
Ideally, the highest concentration should be at least 10 times the Kd. Calculate the ratio of the highest radioligand concentration used divided by the Kd reported by the program (both in nM or pM). The ratio should be greater than 10.
Is the Bmax reasonable?
Typical values for Bmax are 10 to 1000 fmol binding sites per milligram of membrane protein, 1000 sites per cell, or 1 receptor per square micron of membrane. If using cells transfected with receptor genes, then the Bmax may be 10 to 100 times larger than these values.
Is the Kd reasonable?
Typical values for Kd of useful radioligands range between 10 pM and 100 nM. If the Kd is much lower than 10 pM, the dissociation rate is probably very slow and it will be difficult to achieve equilibrium. If the Kd is much higher than 100 nM, the dissociation rate will probably be fast, and may result in the loss of binding sites during separation of bound from free radioligand.
Are the standard errors too large? Are the confidence intervals too wide?
Nonlinear regression programs report the uncertainty of the best-fit values for Bmax and Kd as standard errors and 95% confidence intervals. Divide the SE of the Bmax by the Bmax, and divide the SE of the Kd by the Kd. If either ratio is much larger than ~20%, look further to determine why.
Is the nonspecific binding too high?
Divide the nonspecific binding at the highest concentration of radioligand by the total binding at that concentration. Nonspecific binding should usually be less than 50% of the total binding.
Table 7.5.2 Evaluating the Assumptions of Saturation Binding Analysis
Assumption
Comment
Binding has reached equilibrium.
It takes longest for the lower concentrations to equilibrate, so test equilibration time with the lowest concentration of radioligand.
There is only one population of receptors.
See Theory: Comparing One- and Two-Site Models, below.
Only a small fraction of the radioligand binds, therefore the free concentration is essentially identical to the concentration added.
Compare the cpm obtained for total binding to the amount of ligand. If the ratio is greater than 10% at any concentration, this assumption has been violated. Increase the volume of the reaction but use the same amount of tissue.
There is no cooperativity. Binding of a ligand to one binding site does not alter the affinity of another binding site.
Cell prep: see (Kazami, 2019 #1082), PhReT proposal
Radioligand binding study
3월에 MJFF 성공하면 → 그래도 PE후로 기다리니?, ARG도 불필요하지 않니?
3월에 MJFF 실패하면 → PE후로 기다리야겠지. ARG 필요하려나?
Fibroblasts
PARK2 cells: originally obtained by Matsui-san (ARNU) and Uno-san (EONU) from NINDS. → distributed to NS-DDU, and at least Yamaguchi-san (ARNU) and I currently maintain the stocks
NINDS에서 excel을 Download받아놓았음, 약 35명, to do:major variant에 sequencing 한 결과인지?
20210602 order ID is NHCDR-000840
sPD
cryopreservation
Fibroblast IDs (=submitter tube label)
Subject ID
Gender
age
AAO
Age at Dx
Duration
H&y
race
Submitter_Passage
Submitter_Concentration
box2, Colum1 ack A1, bigger N2 tank DR-430LM, B13F-3140
ND37609
NINDS
NDS00051
M
68
52
65
3
2
Caucasian
3
505555
ND35302
NINDS
NDS00040
M
69
58
58
11
2
Caucasian
2
573333
ND37132
NINDS
NDS00048
M
66
60
60
6
2
Caucasian
2
516666
ND30159
NINDS
NDS00014
F
76
72
74
2
2
Caucasian
2
502000
identifier
Age
gender
Onset age
H&Y stage
MMSE
106-05n
Neonatal
CC-2511
45
GM01650
37
F
GM03652
24
M
ND31618 R42P, homozygous
63
F
44
2.0
28
ND30171 R42P/EX3DEL, Compound heterozygous
54
M
42
2.0
30
ND40078 R275W/R275Q, Compound heterozygous
51
F
46
2.0
ND29369 R275W, Heterozygous
61
F
43
3.0
29
lines 12 ?
pool ; purpose, reason to believe, average anyway
The line is now under expansion culture with around 30 flasks (expected protein is around 9 mg) and if we decide to expand further, the flask number will be around 100.
zone
Disease
Vendor
Number
Gender
Age sampling
Age diagnosis
Age onset
Race
Country
Family History
Genetic Data
Hoehn and Yahr stage
MMSE
3
Healthy control
Coriell
GM01650
F
37
Caucasian
4
Healthy control
Coriell
GM03652
M
24
Caucasian
6
Healthy control
Cell App
106-05n
Neonatal
8
Healthy control
Lonza
CC-2511
F
45
0
Parkinson's disease
NINDS
ND31618
F
63
46
44
Caucasian
USA
R42P
2
1
Parkinson's disease
NINDS
ND30171
M
54
43
42
Caucasian
USA
absent
R42P/EXON3del
2
2
Parkinson's disease
NINDS
ND40078
F
51
47
46
Caucasian
SWEDEN
Maternal aunt
R275W/R275Q cmpd
2
3
Parkinson's disease
NINDS
ND29369
F
61
44
43
Other
DOMINICA
absent
R275W het
3
Disease
Vendor
Number
Gender
Age sampling
Age diagnosis
Age onset
Race
Country
Family History
Genetic Data
Hoehn and Yahr stage
MMSE
Healthy control
Coriell
GM01650
F
37
Caucasian
Coriell
GM03652
M
24
Caucasian
Cell App
106-05n
Neonatal
Lonza
CC-2511
F
45
Park2-PD
NINDS
ND31618
F
63
46
44
Caucasian
USA
R42P
2
NINDS
ND30171
M
54
43
42
Caucasian
USA
absent
R42P/EXON3del
2
NINDS
ND40078
F
51
47
46
Caucasian
SWEDEN
Maternal aunt
R275W/R275Q cmpd
2
NINDS
ND29369
F
61
44
43
Other
DOMINICA
absent
R275W het
3
Uncertain Spans
location
transcription
uncertainty
Q&A table / Comment cells
preserved with verbatim yellow highlighting; the highlight in source overlaps multiple sentences.
low confidence on the exact extent of the highlight in nested tokens.
Fibroblasts inventory / faint-text rows
rows for 106-05a / 106-05n / CC-2511 / C-12302 / GM01650 / GM03652 are rendered in faint grey in the source (likely “deprecated/no-data” rows); preserved verbatim in normal text style.
grey-text styling is dropped in this rendering; row data preserved.
Parkin-PD / ND40078 row / Passage at freeze cells
reads 11 and 6.07 stacked in a single cell in the source; preserved as <br>-separated lines.
low confidence on whether 6.07 is a passage value, mass, or a separate annotation.
Cryopreservation row 1 left column
reads box2, Colum1 ack A1, bigger N2 tank DR-430LM, B13F-3140 reconstructed from partial OCR box2, Colum1 / ack A1, bigger / N2 tank DR-430LM, / B13F-3140.
freezer-location label reconstructed from visible fragments.
Bottom red-banded second copy / zone column
left-most column header zone is partially obscured by the previous row band; preserved verbatim.