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43 Cards in this Set
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FAM171B |
chromosome 2 92 kD poly-Q protein |
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Sub cellular Fractionation |
separates cells into specific organelles |
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Centrifugation |
organelles sediment and rates relative to their physical properties Large molecules form pellet first |
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RCF |
relative centrifugal force multiples of g=1.12x10^-6 r*RPM^2 |
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Mammalian HEK Cells (HEK-293) |
Human Embryonic Kidney
|
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Subcellular Fractionation |
PROTESASES NEED TO BE INHIBITED use protease inhibitors and ice |
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Typsin/EDTA |
Trypsin-Cuts proteins(protease) EDTA-pulls cells off of T-25 |
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ice-cold buffer |
HYPOTONIC (<300 mOsm) cold=inhibits lysosomes makes cells pop |
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Dounce Homogenizer |
Lyses Cells |
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Pellet |
Nuclear Fraction |
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Supernate |
Cytoplasmic Fraction |
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Buffer 2 (RIPA) |
Breaks ALL membranes |
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Protein Assay or Bradford Assay |
determines protein concentration in each fraction ***HAVE TO DETERMINE CONCENTRATION FOR EQUAL LOADING |
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Coomassie Brilliant Blue G-250 |
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 |
measures absorbance absorbance=protein concentration |
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Bovine Serum Albumin (BSA) |
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 |
Separates sub-cellular molecules based on size or molecular weight Charge draws molecules through cell SMALL=Bottom |
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Polyacrylamide and Agarose |
Gels |
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SDS (Sodium Dodecyl Sulfate) |
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 |
Ladder=control |
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Coomasie |
used to stain ALL bands blue in SDS Page |
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Western/Immunoblot |
Identifies PROTEIN OF INTEREST **antibodies only recognize specific proteins |
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FAM171B Antibody
|
injected into rabbit (FAM171B protein) b cells produced in response and made antibodies RABBIT ANTI-FAM171B |
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Nitrocellulose Membrane |
electricity will transfer proteins (100mAmps) |
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Block Buffer |
reduces background INCREASES Specificity of antibody |
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TBS-T |
washes antibodies inappropriately bound |
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Secondary Antibody |
goat-anti-rabbit-IR dye 680 (680 nm light source) HAS DYE |
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Near-Infrared Fluorescence Detection System |
Western Blot |
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Cell Culture |
cells kept alive in virgo allows uniform population of cells |
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HeLa Cells |
derived from malignant cervical tissue |
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HEK Cells |
****Used for cell culture Human Embryonic Kidney |
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DMEM |
media that feeds cells |
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Cell Growth |
37 degres C 5% CO2 |
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PBS/EDTA |
inhibits adhesion proteins cells are removed from T25 |
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Immunofluorescence |
BEST WAY to show where protein of interest is located |
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MeOH |
1. fixes cells into place 2. compromises cell membrane (Ab can enter cell) |
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Block Buffer |
enhances specificity of Ab to target protein |
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Primary Ab (Immunofluorescence) |
rabbit-anti-FAM171B |
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Secondary Ab (Immunofluorescence) |
goat-anti-rabbit-FAM171B-Alexa 568 RED GLOW at 568 nm where FAM171B is located |
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Negative Control Slides |
Not exposed to PRIMARY Ab Makes sure Primary Ab is binding to FAM171B |
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Positive Control |
DAPI Stain of Nucleus BLUE compare if red stain over laps blue stain=nucleus |
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TBS-T (immunofluorescence) |
washes off Ab and rehydrates cells |
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Vectasheild |
Anti-Fade Reagent |