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Enzyme Series Lab 4

Enzyme Series Lab 4. SDS-PAGE Gel Electrophoresis. GOALS. 1. To see how well we purified our enzyme by comparing the crude extract with the purified fraction after electrophoretic separation. 2. To compare how much enzyme our bacteria made before & after IPTG induction.

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Enzyme Series Lab 4

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  1. Enzyme Series Lab 4 SDS-PAGE Gel Electrophoresis

  2. GOALS 1. To see how well we purified our enzyme by comparing the crude extract with the purified fraction after electrophoretic separation.2. To compare how much enzyme our bacteria made before & after IPTG induction.

  3. Reminder: how SDS-PAGE works Sample preparation: Migration of proteins in the gel: _ Negatively charged proteins move toward positive electrode at rates proportional to their MWs. +

  4. Commercial b-gal MW standards - IPTG cells + IPTG cells CE PF 1 2 3 4 5 6 7 8 9 10 Samples to run: • Will load 10µl of each sample

  5. Diluting the CE & PF • Crude Extract & Purified Fractions - Previously calculated the concentrations in mg/ml (no glycerol) • Want 15µg in 15µl (1µg/µl conc.) • Dilute your stock using V1 x C1= V2 x C2 • Will then add 15 µl sample buffer to give 0.5 µg/µl & will load 10 µl on gel (5µg) (If your starting conc. is less than 1µg/µl, must still add sample buffer 1:1 and calculate volume to load that will give 5 µg)

  6. The Electrophoresis Apparatus • Each tank is set up with 2 gels, so two groups will share an apparatus. Be sure to label which gel is which!

  7. Loading the Gel • Use special gel loading pipet tips (long, fine tips). • Place tip at bottom of well and slowly eject the sample, gradually raising the tip as the volume in the well increases. (Steady the pipet using your other hand.) • When all the sample is ejected, draw up a small amount of buffer from the top of the well to smooth out the sample. (Not necessary to wear goggles)

  8. Staining the Gel • Use the green plastic tool to pry apart the two gel plates. • Slide a razor blade under one side of the gel to lift it; gently peel the whole gel off the plate and transfer it to a Tupperware container (label with your initials!). • Cover gel with Coomassie blue for 30 min. with shaking • Pour off stain and add sufficient destain solution (methanol & acetic acid) to cover gel w/ about 1 cm of liquid. Crumple a KimWipe and place it in the container to absorb the dye. • Instructor will photograph gels and post them to the conference.

  9. The finished product • The destaining process washes away any dye that is not bound to protein; dye that is bound to protein creates visible bands. Analyzing the data: • Measure from the bottom of the well to each MW standard band (in cm). Do the same for the bands in the commercial b-gal and PF lanes (CE & cell pellet lanes will have too many bands to do this). • Follow the directions in the lab manual (p. 67) to create a MW standard curve; use the linear regression formula to determine the MW of the bands in the commercial b-gal and PF samples.

  10. Making a MW standard curve • The log of the molecular weight (MW) is plotted versus the electrophoretic mobility (distance traveled from well) of the bands in the MW ladder. • The linear regression equation can be used to calculate the unknown MWs. Distance migrated (cm)

  11. Calculations • Create a standard curve from the markers as directed (use simplified approach). • Calculate MW of b-gal from your gel using the standard curve & compare to known MW of 116 kD for the monomer. Is your enzyme in monomeric or holoenzyme form? Why? • Compare amount of b-gal pre- & post- IPTG induction in whole cells. • Make conclusions about effectiveness of purification procedure and how well your induction worked. • Consider what you could do to verify that a given band on the gel is actually the b-gal protein.

  12. Enzyme Lab Report • Due in lab Monday, March 5 • See grading rubric, lab report instructions in lab manual (p. 76-85) & literature references posted to conference. May also consult resources on scientific writing & Prof. HD’s pet peeves (also on conference). • Each group should make an appointment with me within the next week to go over your data and conclusions.

  13. References to Find Values for Km, Ki, Vmax • For Km & Ki using ONPG as substrate & IPTG as inhibitor: Juers DH, Hakda S, Matthews BW, Huber RE (2003) Structural basis for the altered activity of Gly794 variants of Escherichia coli beta-galactosidase. Biochemistry (42) 13505-13511. • For Vmax values: Wallenfels K (1963) Beta-galactosidase (Crystalline) Methods in Enzymology (5) 617-663.

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