Report Draw a scheme of the GA20OX cloning procedure.

Report

• Draw a scheme of the GA20OX cloning procedure.

Question 1: Why do we study the effect of GA on alapha-amylase in embryoless seeds and not on whole seeds?

Question 2: Volumes

Question 3: Why do we do treatment 4? What is the difference to treatment 3?

Question 4: What is the "debris"?

Question 5: Why is the decrease in absorbance proportional to the quantity of alpha-amylase in the reaction mixture?

Question 6: What do you do if your absorbance in your extract is out of the range of your standard curve?

Western Blot

Steps:

1. SDS-PAGE2. Transfer to membrane: "Blotting"3. Detection of proteins

SDS-PAGE

SDS: sodium dodecyl sulfate

PAGE: polyacrylamid electrophoresis

The goal is to separate proteins according to their sizes.

How would you do that?

Remember

SDS: sodium dodecyl sulfate is a detergent (soap) that can dissolve hydrophobic molecules but also has a negative charge (sulfATE) attached to it

http://www.davidson.edu/academic/biology/courses/Molbio/SDSPAGE/SDSPAGE.html

www.ufs.ac.za

Reductant:

DTT: DithiothreitolB-Mercaptoethanol

The reducing agent beaks any cystine (-S-S-) bonds formed between two cysteine residues

Other stuff in the sample buffer:

Glycerol

Bromphenolblue

How to make the gel?

Polymerization reaction: radical catalyzed reaction

http://www.davidson.edu/academic/biology/courses/Molbio/SDSPAGE/SDSPAGE.html

Polymerization reaction

http://www.davidson.edu/academic/biology/courses/Molbio/SDSPAGE/SDSPAGE.html

Catalysts:

APS: Ammonium persulfate, radical initiator

TEMED:N, N, N', N'-tetramethylethylenediamine, free radical stabilizer

"Discontinuous" PAGE

Low pH (6.8)Low ionic strengthLow Acrylamid concentrationFAST

High pH (8.8)High ionic strengthHigh Acrylamid concentrationSLOW

Visualization of proteins on the gel: Coomasie stain

OR do a western blot

Proteins are transferred to a protein binding membrane.

We will use a nitrocellulose membrane.

Polyvinylidene difluoride (PVDF) is also commonly used.

OR do a western blot