Report Draw a scheme of the GA20OX cloning procedure.

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Presentation transcript:

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-PAGE 2. 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

Reductant: DTT: Dithiothreitol B-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

Polymerization reaction Catalysts: APS: Ammonium persulfate, radical initiator TEMED:N, N, N', N'-tetramethylethylenediamine, free radical stabilizer

"Discontinuous" PAGE Low pH (6.8) Low ionic strength Low Acrylamid concentration FAST High pH (8.8) High ionic strength High Acrylamid concentration SLOW

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