Enzyme Endurance – Student Protocol
Laboratory – Enzyme Endurance
Student Version
Introduction
Each time a boxed section appears in the right hand margin of the text, you must date and initial
that step to certify that you performed it and another team member must date and initial to certify
that the step was verified. Write dates in the following format: day, three-letter month
designation, year (e.g., 13 Nov 06). You may not verify your own work at any step of the
protocol. Entries must be made in blue or black ink.
If you enter data on the worksheet incorrectly, you must use a single line to cross it out, date and
initial it, and enter the correct data with the appropriate “performed by” and “verified by” initials
and dates.
You must document any and all deviations from the protocol, abnormalities, or retests at the end
of the protocol in the comment section. All comments should refer to the specific step or steps
involved as well as specific details of the event along with initials of the team member observing
the event. This also includes any abnormal readings obtained from measurements of any kind
(i.e., pH meter, spectrophotometer).
Enzyme Endurance – Student Protocol
Section 1 – Set Up
STEP
Instructions
1.0
Put on appropriate safety equipment: lab coat,
safety glasses and gloves.
NOTE – If protocol is interrupted and you
must leave immediate area, remove lab coat,
gloves and safety glasses and put on fresh
gloves before restarting.
1.1
Review the experiment and ensure that all
items necessary to complete the protocol are
available.
1.2
Find and assemble all items of labware
listed below.
pH Meter and pH buffer standards
Thermometer
Magnetic stir plate, magnetic stir bars
Scale
Whatman #1 Filter Paper
Tweezers
Microscope slides
Incubator/Oven (370-400)
Spectrophotometer and cuvettes
Beakers
Graduated cylinder
Micropipettors and tips
Stop watch/timer
Vortex
1.3
Find and assemble all chemicals listed below.
(Check MSDS for all chemicals to ensure safe
handling)
Citric acid, FW 192.1
Dibasic Sodium Phosphate, FW 268.07
Peroxidase, from Horseradish
30% H2O2
Guaiacol
Performed By
Date
Initial
Verified By
Date
Initial
Enzyme Endurance – Student Protocol
1.4
Ensure all labware is clean and has been rinsed
with deionized H2O and is completely dry.
Time/Date cleaned
Items are dry yes
no
Enzyme Endurance – Student Protocol
STEP
Instructions
1.5
Check that all scales, stir plates and
spectrophotometer to be used are in working
order. List all equipment to be used below.
Include manufacturer, model number,
calibration date and calibration vendor,
performed by and verification date and initials
required for each item.
1.6
Ensure lab bench or work surface is clear of all
items not pertaining to protocol and has been
decontaminated.
Wash solution used
Time/Date cleaned
Surface is completely dry
yes
no
Performed By
Date
Initial
Verified By
Date
Initial
Enzyme Endurance – Student Protocol
Section 2 – pH Buffer Standards
NOTE – If commercially prepared buffer standards are available they may be used; pH
strips may also be used.
STEP
Instructions
Performed By
Date
Initial
2.0
If using commercially prepared pH buffers
record the data below.
pH 4 buffer
manufacturer
lot number
expiration date
5ml sample taken
date
time
pH 10 buffer
manufacturer
lot number
expiration date
5ml sample taken
date
time
2.1
If using pH strips test both buffer standards
and attach dried strips below.
pH 4 strip
2.2
pH 10 strip
NOTE – If Section 2.0 is not used, Section 2.1
must be used and vice versa.
If a section is not used, mark all empty spaces
with N/A.
2.3
pH meter standardized with pH buffer
standards
calibration date
manufacturer
model number
standardized yes
no
Verified By
Date
Initial
Enzyme Endurance – Student Protocol
Section 3 – Enzyme Buffer Preparation
STEP
Instructions
3.0
Prepare 0.1 M Citric Acid
Weigh out 0.96g citric acid, anhydrous,
FW 192.1
scale calibration date
scale tared
Citric acid:
Manufacturer
lot number
expiration date
amount used 0.96g +/- 0.01g
Dissolve citric acid in 40ml diH20
Addition time/date______________
Stir speed_____________
Hydrated completely
yes
no
Time/date_____________
Bring to final volume of 50ml with
diH20. Label 0.1M citric acid.
3.1
Prepare 0.2 M Dibasic Sodium Phosphate
Weigh out 2.68g dibasic sodium phosphate
(Na2HPO4•7H2O), FW 268.07
scale calibration date
scale tared
Dibasic sodium phosphate:
Manufacturer
lot number
expiration date
amount used 2.68g +/- 0.01g
Dissolve dibasic sodium phosphate in 40ml
diH20
Addition time/date______________
Stir speed_____________
Hydrated completely
yes
no
Time/date_____________
Bring to final volume of 50ml with
diH20. Label 0.2M dibasic sodium
phosphate.
Performed By
Date
Initial
Verified By
Date
Initial
Enzyme Endurance – Student Protocol
STEP
Instructions
3.2
Prepare Citrate-Phosphate Buffer, pH 5.0
Mix 24.3ml 0.1M citric acid with 25.7ml 0.2M
dibasic sodium phosphate and bring final
volume to 100ml with diH20
Obtain pH of buffer
pH
If pH of buffer is not 5.0, adjust with either the
0.1M citric acid solution or the 0.2M dibasic
sodium phosphate solution.
Label citrate-phosphate buffer, pH 5.0.
If using pH strips, attach dried strip below.
3.3
Measure temperature of citrate-phosphate
buffer
22C +/- 5
temperature
Performed By
Date
Initial
Verified By
Date
Initial
Enzyme Endurance – Student Protocol
Section 4 – Enzyme Strip Preparation
STEP
Instructions
4.0
Wearing gloves, measure and cut two 1cm x
2cm Whatman filter paper #1 strips. Handle
with tweezers.
Obtain a microfuge tube of 0.5mg/ml
peroxidase, 1% Bovine Serum Albumin (BSA)
solution. Carefully place the two membrane
strips on a clean glass slide, and spot each strip
with 20µl of the solution. Spot over the entire
surface of the strip if possible. DO NOT
TOUCH WET STRIPS. Use tweezers
whenever handling strips.
4.1
Place strips in oven to dry. Temperature must
be between 37-40C.
4.2
Closely monitor drying process.
Check for strip dryness every 15 seconds.
Use tweezers if it is necessary to touch strip
when checking for dryness. Drying may take
only 1–2 minutes depending on oven
temperature. Strips must be completely dry.
drying start time
drying end time
strips are completely dry
yes
no
4.3
peroxidase addition time
filter paper drying end time
difference of two times
difference of two times is less than one
hour
yes
no
4.4
Place strips in a sealable plastic bag and seal
shut OR proceed directly to Enzyme Activity
Assay.
Performed By
Date
Initial
Verified By
Date
Initial
Enzyme Endurance – Student Protocol
Section 5 – Clean Up
STEP
Instructions
5.0
Discard remaining buffer
amount discarded
5.1
Clean all labware, equipment and surfaces.
Performed By
Date
Initial
Verified By
Date
Initial
Enzyme Endurance – Student Protocol
Section 6 – Comments
STEP
Instructions
6.0
Record all abnormalities, retests, and
deviations
Performed By
Date
Initial
Verified By
Date
Initial
Enzyme Endurance – Student Protocol
Section 7 – Audit
STEP
Instructions
7.0
Ensure all pages are complete
7.1
Ensure all pages are in order
7.2
Ensure all initials and dates are present
7.3
Ensure all non-applicable sections are marked
with N/A
7.4
Ensure Section 5.3 is complete and difference
between peroxidase addition time and strip dry
time is less than 1 hour
7.5
1st Auditor
signature
date
Performed By
Date
Initial
2nd Auditor
signature
date
Time and date documentation turned into QA inspector (instructor)
Verified By
Date
Initial
Enzyme Endurance – Student Protocol
Section 1 – Enzyme Activity Assay
STEP
Instructions
1.0
Turn on spectrophotometer and allow
instrument to warm up at least 10 minutes
manufacturer
calibration date
Set wavelength at 500nm
yes
no
To prepare 10mM H2O2: add 11µl 30% H2O2
to 10ml diH2O. Label 10mM H2O2.
To prepare 25mM guaiacol: add 2.8µl
guaiacol to 10ml diH2O. Label 25mM
guaiacol.
Blank instrument with a solution of
5ml citrate-phosphate buffer
2ml 10mM H2O2
1ml 25mM guaiacol
1.1
Place one enzyme strip in a small beaker with
5ml of citrate-phosphate buffer. Gently vortex
for entire 2 minutes.
strip added to buffer time
strip removal time
1.2
Transfer enzyme/buffer solution to a test tube.
In an additional test tube, mix
2ml 10mM H2O2
1ml 25mM guaiacol
Add H2O2 and guaiacol mixture to enzyme/
buffer solution and immediately place in a
cuvette.
1.3
Place cuvette into spectrophotometer and read
Abs at 500nm over 2 minutes every 15
seconds. Record results below. Each reading
needs a time entry, performed by and verified
by, initials and date.
Performed By
Date
Initial
Verified By
Date
Initial
Enzyme Endurance – Student Protocol
STEP
Instructions
1.3a
Performed By
Date
Initial
Time
Reading
Time
Reading
Strip 1
Strip 1
Strip 1
Strip 1
Strip 1
Strip 1
Strip 1
Strip 1
Strip 1
Color
1.3b
Strip 2
Strip 2
Strip 2
Strip 2
Strip 2
Strip 2
Strip 2
Strip 2
Strip 2
Color
1.4
Chart observations and attach as last page of
this protocol. Use an Excel spreadsheet or
other data management application. Show raw
data with headings and a graph with x and y
axes labeled at set intervals.
Verified By
Date
Initial
Enzyme Endurance – Student Protocol
Section 2 – Clean Up
STEP
Instructions
2.0
Discard remaining buffer
amount discarded
2.1
Clean all labware, equipment and surfaces.
Performed By
Date
Initial
Verified By
Date
Initial
Enzyme Endurance – Student Protocol
Section 3 – Comments
STEP
Instructions
3.0
Record all abnormalities, retests, and
deviations
Performed By
Date
Initial
Verified By
Date
Initial
Enzyme Endurance – Student Protocol
Section 4 – Audit
STEP
Instructions
4.0
Ensure all pages are complete
4.1
Ensure all pages are in order
4.2
Ensure all initials and dates are present
4.3
Ensure all non-applicable sections are marked
with N/A
4.6
Ensure that strips were soaked for 2 minutes at
Section 1.1
4.7
Ensure that absorbance readings were taken
every 15 seconds for 2 minutes at Section 6.3
4.8
1st Auditor
signature
date
Performed By
Date
Initial
2nd Auditor
signature
date
Time and date documentation turned into QA inspector (instructor)
Verified By
Date
Initial