Biol 320 Lab Exam

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FAM171B

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chromosome 2

92 kD

poly-Q protein

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Sub cellular Fractionation

Back 2

separates cells into specific organelles

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Centrifugation

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organelles sediment and rates relative to their physical properties

Large molecules form pellet first

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RCF

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relative centrifugal force

multiples of g=1.12x10^-6 r*RPM^2

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Mammalian HEK Cells (HEK-293)

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Human Embryonic Kidney

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Subcellular Fractionation

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PROTESASES NEED TO BE INHIBITED

use protease inhibitors and ice

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Typsin/EDTA

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Trypsin-Cuts proteins(protease)

EDTA-pulls cells off of T-25

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ice-cold buffer

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HYPOTONIC (<300 mOsm)

cold=inhibits lysosomes

makes cells pop

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Dounce Homogenizer

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Lyses Cells

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Pellet

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Nuclear Fraction

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Supernate

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Cytoplasmic Fraction

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Buffer 2 (RIPA)

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Breaks ALL membranes

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Protein Assay or Bradford Assay

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determines protein concentration in each fraction

***HAVE TO DETERMINE CONCENTRATION FOR EQUAL LOADING

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Coomassie Brilliant Blue G-250

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forms a complex with all proteins

binds and absorbed light at 595 nm

More blue=More Protein

TURNS PROTEINS BLUE

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Spectorphotometer

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measures absorbance

absorbance=protein concentration

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Bovine Serum Albumin (BSA)

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standard curve constructed with known concentrations of BSA

**Calculated amount needed to get 50 ug of each cell extract

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SDS Page/Gel Electrophoresis

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Separates sub-cellular molecules based on size or molecular weight

Charge draws molecules through cell

SMALL=Bottom

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Polyacrylamide and Agarose

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Gels

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SDS (Sodium Dodecyl Sulfate)

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1. coats proteins with negative charge run toward anode

2. denatures proteins unfolds them to be linear

In buffer and gel

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Running Buffer

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Ladder=control

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Coomasie

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used to stain ALL bands blue in SDS Page

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Western/Immunoblot

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Identifies PROTEIN OF INTEREST

**antibodies only recognize specific proteins

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FAM171B Antibody

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injected into rabbit (FAM171B protein)

b cells produced in response and made antibodies

RABBIT ANTI-FAM171B

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Nitrocellulose Membrane

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electricity will transfer proteins (100mAmps)

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Block Buffer

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reduces background

INCREASES Specificity of antibody

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TBS-T

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washes antibodies inappropriately bound

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Secondary Antibody

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goat-anti-rabbit-IR dye 680

(680 nm light source)

HAS DYE

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Near-Infrared Fluorescence Detection System

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Western Blot

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Cell Culture

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cells kept alive in virgo

allows uniform population of cells

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HeLa Cells

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derived from malignant cervical tissue

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HEK Cells

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****Used for cell culture

Human Embryonic Kidney

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DMEM

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media that feeds cells

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Cell Growth

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37 degres C

5% CO2

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PBS/EDTA

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inhibits adhesion proteins

cells are removed from T25

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Immunofluorescence

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BEST WAY to show where protein of interest is located

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MeOH

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1. fixes cells into place

2. compromises cell membrane (Ab can enter cell)

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Block Buffer

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enhances specificity of Ab to target protein

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Primary Ab (Immunofluorescence)

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rabbit-anti-FAM171B

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Secondary Ab (Immunofluorescence)

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goat-anti-rabbit-FAM171B-Alexa 568

RED GLOW at 568 nm where FAM171B is located

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Negative Control Slides

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Not exposed to PRIMARY Ab

Makes sure Primary Ab is binding to FAM171B

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Positive Control

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DAPI Stain of Nucleus

BLUE

compare if red stain over laps blue stain=nucleus

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TBS-T (immunofluorescence)

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washes off Ab and rehydrates cells

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Vectasheild

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Anti-Fade Reagent