Biology 2382B Lecture Notes - Lecture 14: Hoechst Stain, Transmembrane Protein, Primary And Secondary Antibodies
5 May 2018
School
Department
Course
Professor
Lecture 03: Isolation and Analysis of Cell Organelles and Molecules
Labeling Live Cells
- Whe e do iuofluoresee staiig, atiodies a’t ross eraes, so e kill the ells ad
fix them so that the antibody can enter and detect intracellular contents. How can we label live cells?
- 2 ways to label live cells:
1. Antibodies made against specific cell surface proteins can be linked to fluorophores
- primary and secondary antibodies can recognize and a piece of transmembrane protein that
is on outside of the cell, we link these antibodies w/fluorophores
2. Membrane permeable fluorescent dyes can be used to label intracellular structures
- ex. hoechst stain binds DNA in nucleus
- Cells w/bound antibodies or that have taken up the dyes can now be sorted and counted
Fluorescent Activated Cell Sorting (FACS)
- used to sort cells based on structure (i.e. fluorescence) struture = patter of traserae
proteins or pattern of protein structures within cell
1. Live cells pass single file through a laser light beam
2. Both fluorescent light that is emitted and scattered in response to the laser beam is measured by
detectors (quantify amount of fluorescence - below)
3. Individuals cells are forced through a nozzle and given a charge proportional to the degree of
fluorescence detected (cells w/more fluorescence have greater charge)
4. Cells w/different charges are separated by an electric field created by electric plates and collected
(cells w/no charge are discarded, and cells w/greater charge/fluorescence are collected)
Quantification of Cells Expressing 2 Different Cell Surface Markers by FACS
- Lymphocytes/T-cells express the transmembrane proteins Thy1-2 (antibody attached conjugated
w/green fluorophores) and CDC3 (antibody attached conjugated w/red fluorophores)
- As these cells pass through the FACS machine, the intensity of green and red fluorescence emitted by
each cell is recorded...
- T-cells express high levels of Thy1-2 and CDC3 so they
fluoresce high levels of green and red
- The proportion of each cell population can be calculated
(what % of cells have high levels of Thy1-2 and CDC3 can
be calculated)
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Lecture 03: isolation and analysis of cell organelles and molecules. Whe(cid:374) (cid:449)e do i(cid:373)(cid:373)u(cid:374)ofluores(cid:272)e(cid:374)(cid:272)e stai(cid:374)i(cid:374)g, a(cid:374)ti(cid:271)odies (cid:272)a(cid:374)"t (cid:272)ross (cid:373)e(cid:373)(cid:271)ra(cid:374)es, so (cid:449)e kill the (cid:272)ells a(cid:374)d fix them so that the antibody can enter and detect intracellular contents. Cells w/bound antibodies or that have taken up the dyes can now be sorted and counted: antibodies made against specific cell surface proteins can be linked to fluorophores. Primary and secondary antibodies can recognize and a piece of transmembrane protein that is on outside of the cell, we link these antibodies w/fluorophores: membrane permeable fluorescent dyes can be used to label intracellular structures. Ex. hoechst stain binds dna in nucleus. Quantification of cells expressing 2 different cell surface markers by facs. Lymphocytes/t-cells express the transmembrane proteins thy1-2 (antibody attached conjugated w/green fluorophores) and cdc3 (antibody attached conjugated w/red fluorophores) As these cells pass through the facs machine, the intensity of green and red fluorescence emitted by each cell is recorded
