Lyse cells in detergent --> SDS PAGE against MWM to seperate proteins

Separated proteins are transferred out of the gel to the surface of a membrane

Membrane is exposed to Ab specific to the target protein , excess Ab washed off

Binding of Ab is detected using radioactive or chemical tag or coupled to an enzyme that catalysed a chromogenic or luminescent reaction e.g. alkaline phosphatase (could be via a secondary Ab that binds the primary Ab) --> intensity of band proportional to the amount of target protein

Cells, proteins DNA etc can be labelled with fluorescent markers

If a fluorescent marker is bound to a cellular component, the fluorescence intensity will ideally represent the amount of that particular flow cell component

Cell counting

Cell sorting

Determining cell characteristics

Biomarker detection

Operator predetermines or selects certain parameters on how cells should be sorted

Cell sorting machine imposes an electrical charge on each cell so that cells will be sorted by charge (using electromagnets) into separate vessels upon exiting the flow chamber

Specific area of a cell or tissue is photobleached by intense laser light, removing fluorescence from this area

Fluorescence in the bleached area will slowly recover as bleached fluorophores move out and healthy fluorophores from other areas move in

Speed of fluorescence recovery of the bleached area is directly proportional to the diffusion speed of fluorescent or labeled molecules --> estimate diffusion coefficient of the labelled molecule

Sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adosrption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA)

After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen

The detection antibody can be covalently linked to an enzyme or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation

Between each step, the plate is typically washed with a mild detergent olution to remove any proteins or antibodies that are non-specifically bound

After the final wash step, the plate is developed by adding an enzymatic substrateto produce a visible signal, which indicates the quantity of antigen in the sample

Two Abs targeted to two proteins or a protein and a modification (e.g. Ub, pTyr, Sumo etc.)

Abs have attached oligonucleotides

If the two ABs are in close proximity, the oligonucleotides ligate --> rolling circle replication --> detectable signal --> easy readout of PPIs and PTMs

Microarrays consist of a small, solid support (usually a glass slide) onto which DNA molecules are fixed in a grid-like pattern, each spot on the grid represents a different gene.

These arrays are used to measure the expression levels of large numbers of genes simultaneously

Labeling of iPSC Candidates: To visually track the integration and contribution of iPSCs in developmental processes

• Utilize Green Fluorescent Protein (GFP) to label iPSC candidates --> allows for real-time visualization and subsequent analysis of cell integration and behavior in the developmental context

Microinjection into Blastocysts: Carefully microinject the GFP-labeled iPSCs into a host blastocyst

After microinjection, the blastocysts should be cultured under conditions that support embryonic development

Tracing and Confirmation: Confirm that the GFP-labeled iPSCs integrate into the host tissue and contribute to various cell types across the developing embryo; pluripotency of iPSCs is confirmed if they can contribute to tissues in all three germ layers

• Utilize fluorescence microscopy to observe the distribution and intensity of GFP expression across different tissues and developmental stages