Cell Bio (Rice) LOs Class 3

Homogenization

Break cells apart

Subcellular Fractionation

Separate and Isolate components

Types of Cellular Homogenization

Mechanical Shear (Blender)

Sonication (Ultrasonic Vibration)

Solubilizing Cell Membranes (Detergent)

Centrifugation

Separate into fractions

Differential Centrifugation

Components separate by size/density

Low speed separates bigger organelles

Faster for longer separates smaller organelles

SDS-PAGE (Polyacrylamide Gel Electrophoresis)

Separate proteins in gel matrix by size/mass

Larger proteins get stuck in gel matrix

Smaller proteins move with through gel

Immunoblotting/Western Blotting

Move separated proteins from gel to a membrane

Wash membrane with antibody against protein of interest

Detect antibody with chemiluminescence

Genetic Techniques

They allow us to

Add genes

Mutate/change genes

Manipulate gene expression

Add Plasmid to Cell

Include drug-resistance gene in plasmid, only cells with plasmid survive

Creates stably transformed cell lines

Transgenic Organisms

Add plasmid drug-resistant genes to ES cells

Inject ES cells back into blastocyst

Progenies are chimeric

Embryonic Steam (ES) Cells

Inner cell mass of blastocyst

Straightforward to isolate & propagate in culture

Grow indefinitely as pure stem cell population

CRISPR - Clustered Regularly Interspaced Shore Palindromic Repeats

DNA sequence region in bacteria genomes

Enabled acquired immunity in bacteria

Cas

CRISPR associated protein

How does CRISPR work

1. Once infected, bacteria copy & paste viral sequence into CRISPR locus

1. RNA generated from CRISPR locus binds Cas protein & viral DNA

2. Cas protein cuts viral genome

3. Can program the CRISPR-Cas to cut target DNA

CRISPR-Cas can now

Cut DNA

Replace DNA sequence

Edit one base to another

Activate/repress transcription

Modify heterochromatin marks

CRISPR-Cas and Sickle Cell Disease

Turn off the repression of the fetal hemoglobin by using CRISPR-Cas to inactivate the inactivator

Protein-coding genes

DNA transcribed to mRNA

mRNA translates to protein

Protein-non-coding-genes

tRNA & rRNA have a part in translation

miRNA & long non-coding RNA have a part in regulation

miRNA (micro RNAs)

regulate gene expression

cleave complementary mRNA or repress translation

modified by enzyme

cleaved by Dicer

associated with RISC (RNA-Induced Silencing Complex

Immunoprecipitation

Protein mixture incubated with antibody binds to protein of interest & conjugates into insoluble bead

Tests protein-protein interaction because other protein is also indirectly immunoprecipitated