Name Section Lab 03 Enzyme Lab Objectives Safety 1 Measure enzyme activity 2 Determine how changes in substrate and enzyme concentration affect enzyme activity 1 Closed toe shoes must be worn 2 Long hair tied back 3 Goggles must be worn at all times while experiments are being conducted in the classroom Even if you re done 4 Gloves should be worn while performing the experiments 5 All pipet tips should be ejected into the tip waste bin on your bench 6 All ONPG should be poured into the waste container on the side bench Remember to put the lid back on when you are done 7 Rinse your glass cuvettes in the sink and place them in the glass waste box at the front of the room Lab Activity 1 What are the effects of changing substrate concentration on enzyme activity Complete Table 1 and Table 3 on your own before coming to lab Calculate how much ONPG and Z buffer to use for each reaction 1 Plug in a turn on the spectrophotometer at the start of lab so that it has enough time to warm up 2 Dilute the E coli culture 1 5 by pipetting 100 L of E coli culture into a microcentrifuge tube Add 400 L of distilled water close the cap and mix well 3 Clearly label 6 microcentrifuge tubes to correspond with the reactions listed in Table 1 Pipet the diluted Enzyme E coli culture or plain media for the blank into each microcentrifuge tube and then add the appropriate volume of Lysis Reagent Cap the tubes and mix by gentle flicking with your finger Incubate the tubes at room temperature for 10 min 4 While your E coli is lysing clearly label 6 cuvettes to correspond with the reactions in Table 1 Be sure that your label is up near the top of the tube so it will not interfere with absorbance readings 5 Pipet the appropriate volume of Z Buffer into each of the 5 cuvettes and the blank as calculated in Table 1 6 When your E coli are done lysing place the microcentrifuge tubes on ice 7 Quantitatively transfer the contents of each of your lysis tubes to the glass reaction tubes containing the Z buffer What setting should your need on your micropipettor to do this 1 Name Section 8 Add the volume of ONPG stock solution you calculated in Table 1 to each reaction flick gently to mix and incubate at room temperature to begin the reactions Set a timer to count up and record the time when ONPG was added to each reaction tube in Table 2 9 When the reaction turns yellow about the color of a Post It note stop the reaction by adding 500 L of 1M sodium carbonate and record the time you stopped the reaction in Table 2 Gently flick the cuvette to mix and store each cuvette at room temperature until all reactions are complete Complete all reactions before beginning spectrophotometry 10 Be sure to gently wipe off the cuvette with a Kimwipe before inserting the cuvette into the instrument Begin by blanking the instrument with your blank as described in Lab 1 then reading the A420nm for each individual reaction 11 Calculate Gal activity record in Table 2 For more information on this calculation see Lab activity Calculating enzyme activity Lab Activity 2 What are the effects of changing enzyme concentration on enzyme activity and product formation Repeat the procedure from Lab Activity 1 varying the enzyme concentration instead of substrate concentration Complete Table 3 Use 0 4 mg ONPG for all reactions Continue to use 10 L of lysis reagent for all reactions Use E coli culture in the following volumes 3 0 L 10 0 L 30 0 L and 100 0 L Enter your data into Table 4 in a manner similar to Table 2 Once you are finished getting your absorbance readings please turn off and unplug the spectrophotometer 2 Name Section ONPG In Rxn Vol 4 mg mL ONPG Stock Enzyme culture volume Lysis Reagent Z buffer to bring to 1 mL total Concentration of ONPG in Tube M 301 g mol Table 1 Rxn 1 2 3 4 5 Blank 0 1 mg 0 2 mg 0 4 mg 0 8 mg 1 2 mg 0 4 mg 30 L 30 L 30 L 30 L 30 L 30 L media 10 L 10 L 10 L 10 L 10 L 10 L Time started Time stopped A420nm Activity Elapsed time Table 2 Rxn 1 2 3 4 5 Table 3 Rxn Table 4 Rxn 1 2 3 4 ONPG In Rxn Vol 4 mg mL ONPG stock Enzyme culture volume Lysis Reagent Z buffer to bring to 1 mL total 1 2 3 4 Blank 0 4 mg 0 4 mg 0 4 mg 0 4 mg 0 4 mg 3 L 10 L 30 L 100 L 30 L media 10 L 10 L 10 L 10 L 10 L Time started Time stopped Elapsed A420nm Activity time 3 1 Graph the results of each experiment Make sure to title your graph and label the axes The y axis will be activity and the x axis will be either substrate concentration or enzyme volume Do not fit the plot to a line or perform regression analysis Name Section 2 The molecular weight of ONPG is 301 g mol Calculate the molar concentration of substrate in each reaction You should construct a table Show your work 3 How do changes in substrate concentration affect product production and enzyme activity 4 How do changes in enzyme concentration affect product production and enzyme activity Did you observe any limits to activity 5 Are there any problems with the experimental set up for determining enzyme activity How reproducible are the results try comparing the variability of results within your lab What besides technical error could account for this variability and how might one control for any extra variables Paste your graphs onto additional pages in this document along with an image of your calculations 4
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