Ellyn Daugherty SM Biotech Career Pathway BiotechEd
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Transcript of Ellyn Daugherty SM Biotech Career Pathway BiotechEd
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Ellyn DaughertySM Biotech Career Pathway
www.BiotechEd.com
[email protected] www.sargentwelch.com/biotech
www.emcp.com/biotech
Direct ELISA of Alpha-Amylase
Welcome to my workshop:
Fill in the card in the tri-fold brochure (if I don’t already have one from you) and turn it in to me a copy of my curriculum.
Please read through these slides (on the handout in your pee-chee folder).
When done, please read through the lab packet (in the pee-chee folder).
:-) Ellyn
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In Biotech, companies find a product of interest (amylase) and make sure to have assays to show
it’s presence, activity, & concentration
Amylase is an enzyme that catalyzes starch digestion.
It is used commercially in several ways including:
1)Remove starch in products2)Produce sugar from starch3)Processing of cellulostic biofuel
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The rAmylase Project is the focus of the 2nd semester of the SMBCP
• Learn how to assay for a protein of commercial interest (amylase)
• Transform cells to produce that protein (using pAmylase and C2523 cells)
• Scale-up cells to volumes where amylase can be purified and assayed
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The San Mateo Biotechnology Career Pathway
ELISAs taught in Biotech 2 (Ch 6) and Biotech 4 (Ch 14)
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The rAmylase Project Model of rDNA/protein Business
< Chapters 1-5 Basic SLOP
< Lab 5f Amylase PAGE (revised)
< Lab 6e Amylase Producing Bacteria
< Lab 6c Amylase Activity Assay (revised)
< Lab 6d Amylase ELISA & Western Blot (new)
< Labs 7a/7b/7f/7g Amy Spectrophotometry
< Lab 8b Restriction Digestion of pAmylase
< Lab 4j/8b/8g DNA Gel Electrophoresis (revised)
< Lab 8c pAmy Transformation of E. coli
< Lab 9c/9d Amy Ion-Ex Chromatography
< Lab 8g/4h Genomic & plasmid DNA Isolation
< Lab 13i Amylase Gene PCR (new)
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Introduction to ELISA ELISA – short for
enzyme-linked immunosorbant assay
An ELISA uses antibodies with conjugated enzymes to specifically identify and measure the amount
(concentration) of a protein in a solution
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ELISA •A specific antibody (Ab) recognizes a specific protein (Ag).
•An enzyme bound to the Ab causes a color change in a substrate (so you can “see” the Ab is there).
•The more color in a well, the more Ab-enz complex is bound to target protein = higher target protein concentration.
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Di
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Lab 6d Amylase Elisa Kit Expected Results
<<<<< The Standards should look like this …
…and the unknowns should look something like this >>>
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Automated ELISA Plate Reader
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