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Experiment 1.4 Trypsin Digestion and Bandshift
(version 01/21/00)

Background

Biomolecules of different sizes and charges travel at different rates through an agarose gel.  Generally, a smaller DNA molecule will travel further down the gel than a larger DNA molecule.  This also applies to a DNA molecule and that DNA molecule bound to a protein.  The DNA/protein complex will travel slower than the DNA molecule due to the increased size of the complex.  The change in band migration when DNA is bound to protein is known as the "bandshift".  In this lab, we will use a bandshift experiment to test the ability of our purified protein to bind a short sequence of DNA.

                        5'CTGCTATAAAAGGCTG 3'
                        3'GACGATATTTTCCGAC 5'

This DNA molecule has been shown to bind TBP in vivo.  If our purified TBP does bind DNA, we should expect the DNA band to shift upon addition of protein.

To aid in the visualization of the bands, the DNA used in this lab has fluorescein attached to the 3í end of each strand.  Fluorescein is a fluorescent molecule that emits light at 520nm.  This causes DNA bands to glow green when visualized under UV light.  The fluorescein label does away with the need for other visualization methods, such as ethidium bromide staining.
 
 
 Trypsin Digestion of hTBPc

Digestion of the purified protein with trypsin will remove the histidine tag from the N-terminal end of the protein.

1. Prepare a PAGE Gel according to the protocol given under Analytical Techniques on the course syllabus page.
2. Pipette 300 microliters of the pure hTBPc sample into an eppendorf tube.  Using the protein concentration calculated last week, add enough trypsin solution (5 micrograms/milliliter in 1mM CaCl2) to obtain a total trypsin concentration of 0.5% compared to hTBPc.
3. Digest the sample for 30 minutes at room temperature.  At 10, 20 and 30 minutes into the digestion, remove 35 microliters from the digestion and place in eppendorf tubes labeled 10, 20 and 30.  Also prepare a tube with 35 microliters of uncut protein.
4. TLCK is an irreversible inhibitor of trypsin.  After the 30 minute digestion, add TLCK to a final concentration of 1mM to fully inhibit the trypsin.
5. Add 60 microliters of Laemmli buffer to each labeled tube, boil for 3 minutes, and load each sample onto the PAGE gel.  Run the gel at 25 mV for 45 minutes.
6. Stain the gel according to the silver staining protocol in the Analytical Techniques section.
 

DNA Bandshift

7. Prepare a 1% agarose gel according to the protocol given under Analytical Techniques on the course syllabus page.
8. Your TA will provide you with a sample of 0.05mM DNA.  Prepare seven eppendorf tubes with 5 microliters of DNA in each.  This volume corresponds to 2.5 micrograms or 0.25 nanomoles of DNA.  Label the tubes 1 through 7.
9. Using the protein concentration determined last week by Bradford analysis, calculate how many microliters of your protein are needed to obtain 0.25 nanomoles.  The molecular weight of TBP is 21,000 g/mole.
10. Tube #1 will be the control sample, containing only DNA.  Tubes #2 through #7 will contain DNA and protein in varying ratios.  Using your known protein concentration, calculate the appropriate volume of protein for each tube #2 through #7.  Note the total volume (in microliters) of each tube in the last column.

DNA:protein (moles) DNA:protein (microliters) Total Volume

Tube #2      1 : 0.5          2.5 :  ________         _____
Tube #3      1 : 1            2.5 :  ________         _____
Tube #4      1 : 2            2.5 :  ________         _____
Tube #5      1 : 5            2.5 :  ________         _____
Tube #6      1 : 10           2.5 :  ________         _____
Tube #7      1 : 20           2.5 :  ________         _____
 

11.  After addition of the protein to the DNA, let the samples sit at room temperature for 45 minutes.
12.  Add loading dye to each tube, including #1.  The loading dye concentration is 6X, so add enough dye to each tube for a 1:6 dilution.   For a total sample volume of 30 microliters, for example, add 6 microliters of dye for a final volume of 36 microliters.
13.  Load each sample on the 1% agarose gel that was prepared earlier.  Run the gel at 80 millivolts for 45 minutes.  Visualize the gel on a UV lightbox and sketch the bands in your lab notebook.