Term
| List the general steps (3) in Western Blot |
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Definition
| (1) SDS PAGE for protein separation (2) transfer or proteins from the gel to the membrane (3) hybridization of probes |
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Term
| List the order of the sandwich from negative to positive |
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Definition
| (-)sponge pad, filter paper, gel, membrane, filter paper, sponge pad(+) |
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Term
| importance of the sponge pad? |
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Definition
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Term
| importance of the filter paper? |
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Definition
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Term
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Definition
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Term
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Definition
| nitrocellulose membrane, what proteins are being transferred to |
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Term
| Why do we boil the protein samples before placing them in the gel? |
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Definition
| boiling separates the protein aggregates |
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Term
| What do you do with over-boiled samples? |
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Definition
| toss the proteins because over-boiling causes multiple proteins to aggregate and they will not be usable, the concept is to heat them just to release the double bonds |
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Term
| What is the purpose of the blocking buffer? |
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Definition
| prevents the probe from binding to nonspecific sites on the membrane surface, binds to the membrane leaving the proteins exposed to antibody binding |
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Term
| What enzyme is active in an enzyme reaction system? |
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Definition
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Term
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Definition
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Term
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Definition
| investigates RNA structure and quantity |
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Term
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Definition
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Term
| Why do we wash between antibodies? |
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Definition
| subsequent washings remove excess, unbound antibody |
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Term
| why use a secondary antibody? |
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Definition
| against the first antibody, linked to an enzyme such as alkaline phosphatase is used for detection. The membrane is then incubated in a solution containing phosphatase substrates that yield chromogenic products |
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Term
| In what direction does the gel run and why? |
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Definition
| it runs toward the positive anode (red) because the SDS detergent breaks down the tertiary structure and gives the protein a negative charge. |
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Term
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Definition
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Term
| What is the first step in western blot? |
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Definition
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Term
| What is the polymer for an SDS PAGE gel |
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Definition
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Term
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Definition
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Term
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Definition
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Term
| What are the two parts of SDS PAGE |
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Definition
SDS = sodium dodecylsulfate, detergent that denatures proteins 3D shape
PAGE = polyacrylamide gel electrophoresis, separates proteins based on size |
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Term
| Which labs use Western Blot? |
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Definition
| Microbiology and Serology |
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Term
| General steps of the Western Blot? |
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Definition
denature the protein
perform protein electrophoresis
blot the protein from the gel onto the membrane using the gel-membrane sandwich
membrane placed in blocking buffer
primary and secondary antibody are hybridized to the membrane for detection |
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Term
| how can proteins in western blot analysis be transferred from gel to membrane? |
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Definition
| direct transfer, capillary flow, and electric current |
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Term
| how is western blot sandwich assembled, negative to positive? |
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Definition
| sponge pads, filter paper, gel, membrane, filter paper, sponge pads |
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Term
| protein gels are visualized on SDS PAGE gels with ______; DNA is visualized on agarose gel with ________ |
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Definition
| coomassie blue, Ethidium Bromide |
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Term
| What is western blot used to confirm? |
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Definition
| ELISA results for HIV and HepC |
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Term
| what is the purpose of blotting? |
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Definition
| facilitate probe binding and signal detection |
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Term
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Definition
| capillary transfer (12-15 hours), electrophoretic transfer (electric current) |
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Term
| which node is above the gel? |
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Definition
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Term
| which node is below the gel? |
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Definition
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Term
| two methods of electrophoretic transfer |
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Definition
| tank method (large proteins) and semidry method (small proteins with gel-membrane sandwich) |
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Term
| 5 steps for electrophoresis transfer |
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Definition
| 1. prepare transfer buffer 2. equilbrate gels and membrane 3. assemble gel and membrane sandwich 4. set up transfer cell 5. start the transfer |
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Term
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Definition
| required to prevent probe from binding to nonspecifc sites on membrane |
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Term
| why does the membrane have to be incubated in the same buffer? |
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Definition
| makes it miscible with future antibody solutions |
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Term
| What is the probe for southern and northern blots? |
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Definition
| single stranded fragment of nucleic acid which will hybridize specifically with the target DNA or RNA |
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Term
| What are the probes for western blots? |
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Definition
| specific binding proteins or antibodies |
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Term
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Definition
| binds to proteins causing them to unfold, forming linear structures, disrupts multi protein subunit interactions with lipids and contains sulhydral reducing agents to break dilulfide bonds between polypeptide chains |
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Term
| What is the purpose of boiling the samples? |
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Definition
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Term
| which direction is PAGE run? |
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Definition
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Term
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Definition
| smaller polypeptides the higher the gel concentration |
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Term
| what is important about acrylamide? |
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Definition
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Term
| how are proteins visualized? |
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Definition
| coomassie blue or silver stain |
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