Teaching Points for Protein Characterization Lab

VDS Staff,

There are 2 days to the lab

PART 1: prepare samples (this could be done in advance and frozen – i.e. previous day)

PART 2: run gel and stain gel

Destain overnight

PART3: Image destained gel

Dry gel on dryer and take another image

REAGENTS:

The Blue loading dye: use the one for protein, not the DNA loading dye!  The glycerol will make it sticky – so they should cut their pipette tip with scissors if necessary.

We have used Imperial Protein stain and fresh.

We can use the fresh stuff – but they need to save it when they are done (don’t dump Imperial Protein stain down the drain)

IMPORTANT CONCEPTS & SKILLS:

You will have to demonstrate how to clear out the wells with a syringe first.

You will really have to watch  them load their samples.

When there gel is done running – they won’t be able to see any bands (only the ladder) – ask them what was wrong with their gel? They will freak out – but when they stain it – they will see their bands within an hour!

 

That bigger proteins will be at the top of the gel

This is a denaturing gel – so the proteins are NOT in their native conformation – but rather unraveled

 

Explain the different lanes to them and what they ‘should’ see

Ladder – comes from known proteins of known sizes

Flow Through – should have lots of soluble protein (many bands or a smear)

Wash – should have a little bit of protein show up

Elution 1 – ideally – one sharp band

Elution 2 – no bands

 

Be sure to help them see the relationship between their Nanodrop readings from the previous lab and their lanes on the gel. They should see a correlation (ie – if their band is stong – they should have a high protein concentration reading)

 

Drying the gel – we are basically removing the water and leaving the polyacrylamide mesh behind.

 

TROUBLESHOOTING:

everything

 

The notebook for this one is not due this week – but the next . This is so that they can use the labnotebook to write their report.

 

However, the will need to Post their Images from the lab to the Wikispaces this week