20240722_184138

20220518 아래 platemap 보면 5.4mg 로 되었음.

3H-BCPP-EF	1nM
Rotenone block	1 uM
Self block (cold probe)	BCPP-EF 10 uM
Protocol: incubate with protein addition for 1h → remove filters → incubate in scintillation fluid for 4+ hours and count by liquid scintillation counting.	TNT-12465 plate map: Healthy Fibroblast (GM01650), DMSO (n=1, n=2, n=3), Rotenone 1uM (n=1, n=2, n=3), BCPP-EF 10uM (n=1, n=2, n=3) across columns 1–9, with rows A–F highlighted yellow; row F additionally tagged "[3H]BCPP-EF 1nM filter contr"; row H "input" at columns n=1, n=2, n=3; "input count 10nM 3H BCPP-EF 溶液을 10uL スポット して乾燥"; right-side annotation "200ug / 400ug ([3H]BCPP-EF 1nM)" with "Protein 1.8mg / 3.6mg / total 5.4mg"; "Well 당 (이래와 같이) 1,000 uL 넣는 듯".

Wells	Buffer	Blocker	Radioligand	Buffer	Protein
Total	100	—	100	700	100
Non-specific	—	100	100	700	100

[Filter blank wells], filter 안 타는 듯. add everything (add 100ml of buffer instead of protein) except protein. 즉 Replace protein with equivalent volume of buffer. This will tell us what the background signal of the radioligand binding to the filter is. We want this to ALWAYS be below the signal in the wells.

[input radioligand] filter안 안 타는 듯. as well, I just want to be clear that the amount of radioligand added per well only needs to be counted by liquid scintillation counting and does not need to be filtered.

Result interpretation	AK: • I find it concerning that doubling the amount of available target at a constant radioligand concentration is not doubling the number of counts. No explanation comes to mind here—are the two concentrations being prepared differently? Were they from different flasks for example? As I've mentioned before, the number of counts seems low still,
Future direction

Saturation analysis-only HC (GM01650)

May 20, 2022		homogenate
lines		- HC GM01650,
concentration	Protein	400 ug,
3H-BCPP-EF		10 conc (ie 40nM, 25nM, 13nM, 7nM, 5nM, 3nM, 2nM, 1nM, 0.7nM, 0.5nM), ie ☐ Clusters concentrations around what would be the linear phase of the curve and hits a couple points that should be at the higher plateau 20220524 AK RE-calculation: 50, 35 18.6 10.7 7.3 2.4 1.2 0.6 0.5 0.2
Rotenone block		안힘 (이유: in PM tissue ARG, the block with rotenone was poor . so I would use the self-block for this saturation curve.)
Self block (cold probe) protocol		BCPP-EF 10 uM TNT-12465 plate map: Healthy Fibroblast (GM01650), 400 ug/well; [3H] BCPP-EF [nM] 40.000, 25.000, 13.000, 7.000, 5.000, 3.000, 2.000, 1.000, 0.700, 0.500, 0.000 across plate1 columns 1–11 with rows A–F yellow + green; "3H BCPP-EF 1nM wash 2mL—図"; right-side annotation "DMSO" / "BCPP-EF 10uM"; second plate2 row "[3H] BCPP-EF [nM] 400.000, 250.000, 130.000, 70.000, 50.000, 30.000, 20.000, 10.000, 7.000, 5.000, 0.000" across columns 1–11 with rows A–F orange; "input count 500nM ~ 3H BCPP-EF 溶液을 10uL スポット して乾燥"; "タンパク使用量 26.4 mg". Protein amount: 400ug × 10 conc × (triplicate), x (blocking +/-) = 24mg + prepare 20% extra for pipetting error
Result interpretation		Masato: kd seems to be higher than we expected high affinity site at ~2.5 nM (Bmax ~50 fmol/mg; 2.5nM) low affinity site at ~47nM (bmax ~218 fmol/mg; 10.9nM) → we would have to go up to ~500nM for the assay to saturate. This low affinity site may have a nonspecific component I used 42.5 Ci/mmol in the calculation as an assumed specific activity for the radioligand as this is the SpecAct
Future direction		다음 둘 중 어느 상황인지 가리자: we are seeing 2 sites in fact → then we can focus on the first plateau (ie high affinity site) as the lower affinity site should be a non-issue for tracer concentrations in vivo Or we are still not reaching saturation for a single site.? → Then we need higher radioligand concentrations. [next steps options] Another a more detailed saturation analysis with GM01650] AK: 15 concentrations (0.5, 1, 2, 2.5, 3, 6, 8, 10, 14, 18, 25, 40, 75, 100, 200) , Duplicate wells, 4 wells, 0.4mg protein, 15 concentrations (a lot of radioligand?) 24 mg protein (디 radioligand가 부족해서 75 nM까지밖에 못 하는 문제 Concentration response curve to assess inflection points of sigmoidal curve (uses one radioligand conc with varies Cold compound)

Concentration response curve (only HC (GM01650))

design	Concentration response curve to assess inflection points of sigmoidal curve (uses one radioligand conc with varies Cold compound) -radioligand: 4nM 단일 concentration (because the radioligand gives a good window at this concentration), -protein: 0.4mg) -Self-blocker: The final cold concentration shown below Actual plate map: 최저 용량이 0.1nM 아니라 1nM이네? TNT-12465 plate map: Healthy Fibroblast (GM01650), 400 ug/well; cold BCPP-EF [nM] across plate1 columns 1–11 with values 0, 3000, 1000, 500, 300, 100, 50, 30, 10, 5, 3, 1 (n=1 row A, n=2 row B) and 0.5, 0.3, 0.01 (n=1 row C, n=2 row D); "[3H]BCPP-EF 4nM (filter control)" row F; right-side annotation "[3H]BCPP-EF 4nM final"; "0.4mg*2*16=12.8mg"; "タンパク使用量 12.8 mg"; row "input" at column 10.
Results	Two Site Fit: %inhibition vs Concentration (nM); annotated Hi IC50 = 9 nM (Ki ~5 nM), Lo IC50 = ~400 nM. AK: I do not see a well-defined second binding site by concentration (so, low affinity site를 위한 고동도는 더 이상 볼 필요 없겠다.) Because the Ki is 5nM, there SHOULD be saturation at 50nM.
Future direction	그러니, (Nine): 1,3,4,7,9,11,15, 40, 75 filter blank wells for each radioligand concentration as well. This should be n=2 wells for total and nonspecific conditions. to make sure there is no plate based binding at the high concentrations that act to confound our data (possible show up as a second binding site, . they should confirm that the final radioligand concentration will align with the above by counting prior to adding to the assay

Saturation analysis (Main)

2022 5/31

homogenate HC (n=3), GM01650 (이미 했더라도 다시 한번), 106-05n, CC-2511 PKRN-PD (n=4),: ND31618, ND30171 (17.07mg), ND40078, ND29369 (Heteroz) sPD (n=2): ND37132, ND30159 2022/5/27 Cell Note Date Plate GM01650 Healthy fibroblast 220527 plate1 106-05n Healthy fibroblast 220527 plate2 ND31618 PD-PARK2 (ARG42PRO) fibroblasts 220527 plate3 ND37132 Fibroblast of sporadic Parkinson's disease patient 220527 plate4 Input count 220527 plate5 2022/5/30

Uncertain Spans

location	transcription	uncertainty
Top platemap right annotation	Well 당 (이래와 같이) 1,000 uL 넣는 듯	The Korean reading 이래/아래 (likely typo for 아래) is preserved as written in the source.
Saturation analysis 3H-BCPP-EF row	Clusters concentrations around what would be... and the leading icon (☐)	The ☐ at the start of this sub-bullet is rendered as an empty placeholder glyph in the source; preserved as such.
Two Site Fit annotation	Hi IC50 = 9 nM (Ki~5 nM); Lo IC50 = ~400 nM	Annotation box is small inside the embedded plot; “9 nM” reading is consistent across both AppleVision and PaddleOCR.