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20240722_184703

8 min read

Mouse strain

B6C3F1,Hybrid micea cross between female C57BL/6 and male C3H mice.

Transgenic Mouse

  • Generation
    • a- Most transgenic mice are generated by the direct microinjection of DNA fragments into the pronuclei of fertilized eggs. The majority of integration events occur prior to the first round of chromosomal DNA replication. therefore most G₀ transgenic mice are derived from a mosaic embryo.

Fluorescent proteins

Process

  • Genome editing
    • a- Tag by fusion: the precise insertion of these fluorescent protein tags into the genetic material of many diverse organisms

Examples

proteinabsorbs lightemits light
green fluorescent proteins (GFP)이게 군이 아니라 하나의 proteinbetween 540-590 nm550-650 nm
enhanced GFP (EGFP)
mCherrymonomer
Dendra2
monomeric red fluorescent proteins (mRFPs)

Histology

dropout

Assays

WB

  • SDS가 protein 주위를 negative charge 로 둘러싸서 WB 에서 흘러가게 만듬.
  • Mercaptoethanol reduces SS bond (a common covalent bond) ie breaks it.
  • 박민우: SDS 가 SS bond를 다 깬다. 그러나 다른 covalent bond는 유지된다. 그러나 찾아보니 SDS is an anionic detergent. in SDS-PAGE disulphide bridge (-S-S-) in protein(in tertiary structure) is reduced(broken) by β(β)- mercaptoethanol . now SDS binds strongly between them . so both by combined action denature proteins, 그러나 wiki에는 SDS causes denaturation (그러니까 SS bond는 아니고, 3D structure유지하는 Hydrogen bonds는 SDS가 깨나보다).
  • A disulfide bond, also called an S-S bond, or disulfide bridge, is a covalent bond derived from two thiol groups
  • BRIC: (disulfide bond에 의한 결합이 아닌) oligomer에 의한 결합을 확인할 때는 native PAGE라고 하여 SDS를 제거 하고 내리는 젤을 내립니다. 이 경우 SDS가 없기 때문에 단백질의 크기는 물론 단백질 고유의 전하의 영향또한 받게 됩니다. (그래서 basic native, acid 방법이고 유의 영향또한 받게 있는 같습니다) 끊이지 않는다고 해서 oligomer가 유지되는 것은 아닙니다. 참고로 제가 연구하는 단백질은 기본 dimer에 dimer 그 이상까지 형성한다고 알려져 있지만 loading dye (SDS포함하는) 넣고 boiling하지 않아도 boiling한 것과 별반 다를게 없는 결과가 나왔습니다. 물론 SDS가 없는 native PAGE를 하게 되면 dimer, decamer가 나오구요.

결국 monomer 만 남게 되겠네.

[Protein purification]

  1. Cell disruption : by sonication etc.
  2. Solubilisation SDS) can be used to dissolve cell membranes and keep membrane proteins in solution during purification; however, because SDS causes denaturation, milder detergents such as Triton X-100 or CHAPS can be used to retain the protein’s native conformation during complete purification.
  3. Ultracentrifugation: 어떤 게 pellet 으로 남냐면: massive, small, and dense particles

Km: The concentration of substrate at which enzyme functions at half rate. 이게 낮으면 binding (eg enzyme-substrate, protein-ligand) 이 강하다는 얘기.

Isoelectric focusing: used to determine pH of a protein

Dimer: Most dimers in biochemistry are not connected by covalent bonds.. 그럼 WB시 깨지겠네. 그러니까 안 끓이면서 준비하는 Fractionation 에서만 oligomer 보이는 걸테지.

[Joe]

  • Peptide bond: very strong, even resistant to boiling, a form of covalent bonding
  • Hydrone bond: weak

Enzymes required its 3D structurer to be intact to exert it effect.

Protein : 그 안에 amino acid들 끼리는 peptide bond 라 안 끊어지지만, 3D structure 는 Hydrogen bond 로 이어져 있어서 이게 끊어져서 3D structure 를 잃음.

“SDS Stable/ Unstable”. A substance is stable if it is resistant to decomposition or possesses the ability to remain unchanged

Chromatography

Principle

어떤 compound가 자기가 가진 partition coefficient 에 따라 (non-polar) stationary phase 에서 얼마나 머문 후 (polar) mobile phase 로 넘어가는지가 다르다. 이것을 이용해, mixture of compound 를 넣어 각각의 compound 를 separation 시켜낼 수 있다.

Type

Liquid Chromatography:

When a sample is injected, it is adsorbed on the stationary phase, and the solvent passes through the column to separate the compounds one by one, based on their relative affinity to the packing materials and the solvent. The component with the most affinity to the stationary phase is the last to separate.

HPLC (high-performance liquid chromatography

  • an advanced type of LC

  • The difference between traditional LC and HPLC is that the solvent in LC travels by the force of gravity. In the application of HPLC, the solvent travels under high pressure obtained by means of a pump to overcome the pressure drop in the packed column, which reduces the time of separation

  • HPLC requires a pump to inject the solvent

  • Retention time usually represents the x-axis of the chromatogram; however, the y-axis depends on the method used for detection, which is usually a UV detector and measures the intensity of absorbance.

MS (Mass spectrometer)

Principle

  • Molecule/Compound 를 ionization 시켜→ sideforce (ie magnetic field) 주면 compound가 휘는데 (ie separation되는데) (deflect), 그 휘는 정도는 mass 에 의존한다. 그래서 어떤 물질의 mass (the number of carbons 도) 를 알아낼 수 있다. → known mass spectrum fragments (eg. M 14는 CH2) 이용하면 Molecular structure 알아낼 수 있다
  • Hiroshi Sugimoto: requires 50 ul for untargeted LC/MS
  • Native MS: de-naturing 포함.
  • intact protein LC/MS (Kellie, 2021 #2644), good review: relies on a single protein or antibody for capture → forego the digestion step, and the eluate from immunocapture is directly injected for analysis

Output

Peak heights on a mass spectrum are proportional to the number of ions of each mass.

The most intense peak in the spectrum is called the base peak.

Limitation

  • MS alone cannot perform the separation process, when several compounds can have a similar molar mass and fragmentation pattern.

Assay Development LC-MS

Method development (Rate-limiting)Method ValidationMethod implementation
Sample preparation,
Extraction of pure compound
Reference standard (When a reference standard cannot be purchased commercially, custom-synthesized compounds of documented purity can be used.)Acceptance criteria must be established: Specificity and selectivity, Accuracy and precision, LOD--
Calibrator
Protein 의 경우: (protein 은 너무 크므로) select proteotypic peptides (= signature peptide) to represent the target proteins : such peptides are 8-25 amino acids in length,
- Protein quantification using surrogate peptide approach with bioinformatics (sequencing),

The target protein sequence is first compared with the sample matrix proteome → the predicted peptides, unique for the target protein, are identified. (The analysis is based on referably more than one surrogate peptide (one quantitative peptide plus confirmative peptides). protein digestion, using sequence specific proteases, to liberate the predicted peptides from the target protein. (The target protein quantification is then based on monitoring LC/MS signal of the unique target peptides.) →
LC/MS/MS method optimization for quantification of the surrogate peptides
the target protein is enriched (Typical enrichment methods are protein A/G capture of antibodies and antibody capture of proteins (target or tag specific antibodies)., or the background proteome depleted (Typical depletion methods are selective precipitation and solid phase extraction, as well use of different commercial depletion resins) 		i)iii)iv)

Uncertain Spans

locationtranscriptionuncertainty
BRIC long Korean note이 경우 SDS가 없기 때문에 단백질의 크기는 물론 단백질 고유의 전하의 영향또한 받게 됩니다. (그래서 basic native, acid 방법이고 유의 영향또한 받게 있는 같습니다)The middle parenthetical fragment runs across two crop tiles with line breaks rendered ambiguously; the joined reading preserves visible tokens in left-to-right order but the parenthetical group ordering may differ slightly from the source layout.
Assay Development LC-MS table, right edgethe i) / iii) / iv) row markers under Method Validation and Method implementationThe Roman-numeral row markers are visible but the ii) cell is not clearly captured between i) and iii) in this photo; they likely continue in 20240722_184706.
Assay Development LC-MS table, last visible rowprotein standardsThe token highlighted in yellow at the bottom edge is partly cut off; the visible characters read protein standards but the trailing portion of the sentence (spiked at known...) extends below the photo crop.

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