Ellyn DaughertySM Biotech Career Pathway

www.BiotechEd.com

ellyn@BiotechEd.com 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

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

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

The San Mateo Biotechnology Career Pathway

ELISAs taught in Biotech 2 (Ch 6) and Biotech 4 (Ch 14)

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)

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

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.

Di

Lab 6d Amylase Elisa Kit Expected Results

<<<<< The Standards should look like this …

…and the unknowns should look something like this >>>

Automated ELISA Plate Reader

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