Direct ELISA of Alpha-Amylase

Welcome to my workshop:  Ellyn Daugherty SM Biotech Career Pathway

www.BiotechEd.com

[email protected]

www.sargentwelch.com/biotech www.emcp.com/biotech 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 products 2) Produce sugar from starch 3) Processing of cellulostic biofuel

The rAmylase Project

is the focus of the 2 nd 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 0,35 0,3 0,25 0,2 0,15 0,1 0,05 0 -0,05 0 Ion Exchange Chromatography 5

Fractions (#)

10

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 e nzyme l inked i mmuno s orbant a ssay 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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