만약 only a very small fraction of the ligand binds to receptors (or to nonspecific sites).
→ 면 the free concentration of ligand is approximately equal to the concentration you added
This assumption vastly simplifies the analysis of binding experiments, and the standard analysis methods depend on this assumption. ligand depletion 10% 이하 가능성도 커짐
Radioligand depletion is most pronounced at the lowest ligand concentrations (이유 모르겠네)
Future direction
Ashley:
- i) ↓ Radioligand (1-2nM 중에서 1 농도, 근데 Yoshirou는 96well에 자리도 남으니 1&2nM 둘다 하련다)), or ↑ wash number (from 2x 2mL ice cold PBS to 4x 2mL PBS (keeping duration the same))
ii) ↓ protein concentration (so 200, 400, 500 ug, to confirm repeat of the SB in these conditions) - saturation analysis.
더 나중에는
If the counts from the next cell homogenate assay look good, I think we should go forward with a first full saturation curve with 12 points (Just thinking ahead). I will start thinking about the concentrations we should aim for as this should not be doubled concentrations.
3H-BCPP: Simplify (only 6.25, 25, 100 nM)
cell number는 당분간 동일하게 가자
increasing the number of cells
adding ice cold 0.1% BSA to the wash buffer followed by ice cold PBS to try to reduce the non-specific binding that I think can be attributed in part to the MEM buffer containing FBS and sugars.
More future plan: saturation analysis (12 concentrations, in triplicate)
Update 20211210 (Yoshirou, only living cell)
| homogenate | Living cells | |
|---|---|---|
| concentration / Protein / cell | 2 × 10^4 cell/100 ul/well | |
| 3H-BCPP-EF | 6.25, 25, 100 nM | |
| Rotenone block | 1 µM | |
| Self block (cold probe) | 10 µM | |
| protocol | wash (200 ul per wash): 2 vs 4 times | |
| Result interpretation |
Number of washes did not greatly impact specific binding SB increased (20210806보다) Radioligand should reach saturation around 25 nM, (saturation of target is usually achieved by 10-20x the kd concentration of radioligand. This of course leads me to believe we see a lot of non-specific binding still.) Bar chart (% of total CPM, Total binding / Rotenone (10uM) / BCPP-EF (10uM)): • 6.25 nM, 2 Washes: 62% / 23% / 34% — 4 Washes: 53% / 60% / 37% • 25 nM: 4 Washes 42% Some NSB remains (which we can continue to work to reduce) or an additional binding site | |
| Future direction |
• Include wells without cells (reagents only) • Washing cells ahead of assay with 50 mM Tris-Saline buffer to remove MEM (assess whether cells begin dying?) • Increasing cell numbers (limiting due to growth rate) • One small experiment I'd like to propose is the protocol carried out without cells. I want to see how much of this non-specific binding can be attributed to plate binding. We can wait until we get the results from homogenate binding if there is a substantial cost with doing this small assay. • But, Given the data generated by Abbvie for MiNDMaps , showing that BCPP-EF does not show MC1 activity, but only binding to the target, do we believe we should continue to optimize this live cell assay (Pending homogenate data)? | |
Update 20220108 (1st small repeat)
| homogenate Healthy | |
|---|---|
| concentration / Protein | 200, 400 ug |
| 3H-BCPP-EF | 1 nM, 2 nM |
| Rotenone block | 1 µM |
| Self block (cold probe) | 10 µM |
| BCPP-EF | 10 uM |
| protocol | Culture in a large flask → Get homogenate. They conducted the homogenate binding assay after stocking the homogenate once in freezing condition. (We are not sure if this affects the result, but considering the next experiment size and time schedule, it seems to me appropriate to stock the homogenate and run the assay in one day after having all lines.) |
| Result interpretation | Yoshirou: The effect of increased protein concentration was limited. We could confirm a significant signal reduction - specific binding - by both rotenone and self (BCPP-EF cold) blocking (they block with self and rotenone were similar.) Ashley: I will use only the CPM for now to evaluate the results. |
| Future direction | 아래를 하려고 했으나 homogenate 잔여가 없어서 못 하고, 대신 just RI-filtering assay? I mean compare the RI count with and without 3H-BCPP-EF go through the filter. If the 3H-BCPP-EF is sticky to the filter plate, then we can see the difference between those two. Otherwise the count should be similar between them. = Ashley: just doing the filter blank wells with the unfiltered radioligand concentration as well will be good. If they can keep the radioligand concentration very close to that of the most recently reported assay, it would be ideal. |
Planning saturation analysis (PROTEIN amount 400 ug/well)
20220518 expansion outcome → 9 mg/30 flasks (예상이었는데 더 나왔네)
- 9 conc, triplicated well replicates, pooling x: 36 mg/line
- 9 conc, duplicated well replicates, pooling x: 18 mg/line (= 9 mg/line (30 flasks/line)). 계산해보면 400 ug × 9 × duplicates × (blocking +/-) = 14.4 mg, 여기에 extra 25%해서 18 mg 로 된 거구나
- 9 conc, duplicated well replicates, pooling o: 9 mg/line
Cohort:
- HC (n=3), GM01650 (82.78 mg), 106-05n (26.3 mg), CC-2511 (29.85 mg)
- PKRN-PD (n=3): ND31618 (24.9 mg), ND30171 (17.07 mg), ND40078 (27.18 mg)
- sPD (n=2): ND37132 (39.93 mg), ND30159 (amount to come)
2nd repeat to assess counts at 200ug and 400ug: 3.6 mg but I’d include more for error: 4 mg total. → GM01650 (to be expanded to get 22 mg (4 mg for a validation study + 18 mg for a saturation study)
Update 20220215 (RI filter assay)
| homogenate Healthy | |
|---|---|
| concentration / Protein | 200, 400 ug |
| 3H-BCPP-EF | 1 nM |
| Rotenone block | 1 µM (Plate map 보면 없었을 것) |
| Self block (cold probe) | Plate map 보면 (없었을 것) |
| protocol / Result interpretation / Future direction | RI filter blank assay (filter sticky test) |
Update 20220518 (2nd small repeat)
Objective: To work out the variability and the low count
Healthy Fibroblast GM01650
| homogenate Healthy Fibroblast (GM01650) | |
|---|---|
| concentration / Protein | 200, 400 ug, 원래 다음의 논의 있었으나: 3.6 mg but I'd include more for error: 4 mg total. → 20220518 아래 platemap 보면 5.4 mg 로 되었음. |
| BCPP-EF | 1 uM TNT-12465 |
| Rotenone block | 1 µM |
| Self block (cold probe) | BCPP-EF 10 uM |
| protocol | incubate with protein addition for 1 h → move filters → incubate scintillation fluid for 4+ h. Well 당 (아래와 같이) 1,000 µL 넣은 듯. |
plate map (n=1 DMSO / n=2-3 Healthy Fibroblast GM01650 / n=2-3 Rotenone 1 µM / n=2 BCPP-EF 10 uM): see body_r05_c01.jpg.
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| time-stamp | photo time 6:41 PM differs from 6:35 PM of the previous adjacent stem | The session photos resume after a 6-minute break; both readings preserved as visible. |
| 20211210 bar chart percentages | 62%, 23%, 34% etc. | Bar values are reconstructed from a small chart; readings are best-effort. |
| 20220215 plate map | the row contains Plate map 보면 없었을 것 notes | The plate map for this iteration is described in the source as missing; reconstruction reflects what the visible cell text describes. |
Cohort CC-2511 | mass 29.85 mg | The cell line code CC-2511 may be CC-2511 or CC-251 with a trailing digit; preserved as visible. |
| TNT-12465 | TNT compound code | TNT-12465 is rendered with stylized digits; reading consistent with the surrounding plate map context. |