| isothermal titration calorimetry (ITC) | The most quantitative; it's not suited for high-throughput screening because of the large amounts of sample required and long titration times. | |
| BioLayer Interferometry (BLI) | ||
| MicroScale thermophoresis (MST) | ||
Notes column (right side of the same table row group):
- measure the thermal stability of a target protein and the increase in protein melting temperature upon the binding of a stabilizing agent.
- measures the melting temperature of a protein (Tm), which is the temperature at which there is 50% denaturation
- deliverable: melting temperature shift (ΔTm)
- measures changes in the thermal denaturation temperature and hence stability of a protein
- The binding of (low molecular weight) ligands → increase the (thermal) stability of protein → increases the melting temperature from 45 to 53 ie (ΔTm 값이 커짐)
- Denatured protein is unable to bind the radioligand and the protein and radioligand will be separated in the gel filtration mini column
Differential Scanning Fluorimetry curve (figure, evidence-only):
Fluorescence vs Temperature (°C, 20–80) for two traces, DMSO and PF-06928215, with a ΔTm arrow between the two midpoints near 45 °C and 53 °C.
Example of Differential Scanning Fluorimetry of cGAS in the presence or absence of 10 µM PF-06928215.
The thermal denaturation of cGAS is detected via SYPRO Orange dye. The addition of the inhibitor PF-06928215 stabilizes the protein and increases the melting temperature from 45 to 53 degrees Celsius.
(Rosa et al. 2015, PMID 25918038) The cutoff for the ΔTm (5 °C or greater) was derived empirically from inspection of some pathological examples.
SAR (Structure Activity Relationships)
- Definition
- a- to predict biological activity from molecular structure.
- b- This is because similar compounds may have similar physical and biological properties. There is a relationship between molecular structures and their biological activity, and this principle is referred to as Structure Activity Relationship (SAR).
- Tools
- a- CDD Vault
- Applications
- a- risk assessment of uncharacterized compounds, data from the most sensitive toxicological endpoints should be included in the analysis,
Immunoprecipitation
| Immunoprecipitation No boiling, so detects natural form of protein | Co-Immunoprecipitation | |||
|---|---|---|---|---|
| purpose | 한 단백질을 특이적으로 분리 (isolate)(하여 확인) | 두 단백질의 결합을 확인 (eg. p1 + p2) | ||
| process | Sample prep: | protein release | Cell lysis: (Jain 2012, for SiMPull) it is critical to use non-denaturing conditions in order to preserve the physiological interactions that may occur. We avoid SDS and other strong ionic detergents for lysis as they can potentially denature the immobilized antibodies or disassemble protein complexes. | |
| Protein denature | 이 단계에서 heating 이겠지? 이거 하나? | |||
| Protein dissociation | 분해 | |||
| Incubation on the antibody coated surface |
항체와 단백질 1 의 결합 항체-단백질 1 복합체와 A/G agarose bead 와의 결합(시켜 침전시킴) - precipitation (침전) 위해서는 antibody-protein complex 가 충분한 무게를 갖게 해야 한다. 이 역할을 하는 것이 A/G agarose bead 이다. - A/G 는 protein A 와 protein G 를 뜻한다. Protein A/G 는 원래 bacteria 의 단백질로 Protein A 는 Staphylococcus aureus (황색 포도상 구균)에서 발견되었고 Protein G 는 Streptococcus 에서 발견되었다. 이, 단백질들은 모두 bacteria 의 surface protein 이며 항체를 구성하는 IgG (immunoglobulin G) binding domain 을 다수 가지고 있어 IgG 와 강하게 결합하는 단백질이다. Protein G 는 albumin 에도 강력하게 결합하나 immunoprecipitation 에서는 방해되므로 Protein G 의 albumin binding domain 은 제거한 후 사용한다. - 즉, 항체와 결합된 단백질을 침전시키기 위해서 bead 에 protein A/G 가 코팅 되어 있는 것이다. 이것은 antibody 의 IgG 부분과 강력히 결합할 수 있게 하여 유리 구슬이 precipitation 을 할 수 있는 충분한 무게로써 작용한다 |
항체와 단백질 1 의 결합 (eg. antibody to P1 is used) 항체+ 단백질 1+ 단백질 2 복합체와 A/G agarose bead 와의 결합(시켜 침전시킴) | ||
| 3 | Centrifugation (를 통해서 bead 와 단백질 복합체를 모은) → 후 Washing 을 통한 비 특이적인 결합 제거 | |||
| 4 | WB 하여 그 단백질 1 이 맞는지 확인 | WB 하여 그 단백질이 맞는지 확인 (eg. antibody to P2 is used) | ||
Proximity ligation assay (PLA)
| also referred to as Duolink® PLA technology, permits detection of protein-protein interactions in situ (at distances < 40 nm) at endogenous protein levels. |
| single molecule resolution and objectively quantified in unmodified cells and tissues |
Pull-down assay
| similar to immunoprecipitation, except that a "bait" protein is used instead of an antibody. |
| https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/pull-down-assays.html?gclid=CjwKCAjw0N6hBhAUEiwAXab-TXhiXKJJ57vjByd0jlQco0JSMF0WQh1x1E2gdsuxFi9F8Oa6N_RKVxoCtqgQAvD_BwE&ef_id=CjwKCAjw0N6hBhAUEiwAXab-TXhiXKJJ57vjByd0jlQco0JSMF0WQh1x1E2gdsuxFi9F8Oa6N_RKVxoCtqgQAvD_BwE:G:s&s_kwcid=AL!3652!3!386245227784!!!g!!!661761931437046&cid=bid_pca_ppf_r01_co_cp1359_pjt0000_bid00000_0se_gaw_dy_pur_con |
Single molecule Pull-down (SiMPull) assay
- {Jain, 2012 #2290}: protocol and principle, schematic diagram
Near InfraRed counting
- Method: {Sonne, 2021} Fresh-frozen sections (fixation X) → slides → incubation with a regular primary antibody → incubation with a infrared fluorescent secondary antibody → LICOR Odyssey imager
Single Molecular Counting
| Proteina | Klenerman | |
|---|---|---|
| Co-IP | Pull-down, a single-molecule pull-down (SiMPull), SiMPull combines conventional pull-down assay with single molecule imaging: protein complexes are pulled-down directly from | |
| TIRF (total internal reflection fluorescence) microscopy | ||
| What is the fluorophore? | STORM = single molecule fluorescence imaging? enables direct visualization of cellular protein complexes at the single molecule level. Stochastic optical reconstruction microscopy (STORM): one of a family of Nobel Prizewinning super-resolution Single Molecule Localization Microscopies (SMLM) | |
| readout | (absolute) single molecular count | Number of ASC aggregate/FOV |
| OD | ASC: 1000 ASC count (100 cells) enables a statistically meaningful number of events to be measured | ? |
| throughput? | ||
| problem & troubleshoot |
Sampling issue? Poisson noise: uncertainty of the measurement is √n where n is equal to the number of molecules counted. For example, if four molecules are counted, then the uncertainty of the measurement is 4±2, which makes for a CV that is analytically unacceptable. Too low concentration → pre-concentration? non-specific binding (NSB): one must detect three times the standard deviation of the background before one obtains a meaningful analytical result. → troubleshooting: surface modification approaches that attempt to passivate the surface so that it does not attract proteins Nonspecific binding of the analyte molecule to surfaces → consequence: signal loss → troubleshoot: passivate these surfaces and to minimize transferring samples from one vessel to another. Single-Molecule Super-Resolution Imaging: [Single Molecule Localization Microscopies (SMLM) -STORM -Photoactivated Localization Microscopy (PALM) - Ground StateDepletion Individual Molecule Return (GSDIM) | |
| chromophore | Luminophore | Fluorophores |
|---|---|---|
| Examples | aminoluciferin | AMC, AFC, ACC, AMAC, AMCA |
| pNA |
The Neurotoolkit (https://www.alzheimersdata.org/ntk)
- Elecsys ligand binding assay
- Elecsys assay is standardized against LC - MS/MS calibrated
measurements have to be performed in one of the four qualified NTK laboratories: LabCorp Indianapolis (USA), University of Gothenburg (Sweden), Microcoat (Penzberg, Germany) and TrigaS (Habach, Germany).
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
| Pull-down assay URL | long thermofisher URL with gclid=CjwKCAjw0N6hBhAUEiwAXab-...&cid=bid_pca_ppf_r01_co_cp1359_pjt0000_bid00000_0se_gaw_dy_pur_con | The tracking-parameter portion of the URL is small print and the OCR shows two slightly different repeats; transcribed once but the trailing query string may have minor character differences from the source. |
| Immunoprecipitation table, top header row, leftmost cells | three-cell empty header before Immunoprecipitation / Co-Immunoprecipitation | The exact column merge for the leftmost label area (purpose, process, Sample prep, sub-step) is rendered in the photo with three nested header columns; flattened to a 3-cell empty header here. |
Single Molecular Counting table, OD row, Klenerman column | ? | Visible glyph is a single yellow-highlighted ? in the source — kept as-is rather than being treated as missing data. |
Single Molecular Counting table, chromophore row | pNA placed below chromophore and to the left of the Luminophore/Fluorophores row | The mini sub-table in the source overlaps the chromophore label and the Luminophore/Fluorophores row; pNA is preserved as a chromophore example but the exact layout of empty cells is approximate. |