Done By: WB_Silver7 School Name: Al Wakra Independent Secondary School for boys

1.  Ahmad Teyseer Abdulla Alemady
 Tamim Mohammad Hamad

4. Activity
1

5. control Whole Whole
tail
Pieces
Activity
1
Luciferin + ATP + Oxygen → luciferase → photon

6. Tube A B C D
Tube Name Control Whole tail Pieces tail Whole
Enzyme luciferase luciferase luciferase luciferase
Indicator ATP + O2 ATP + O2 ATP + O2 ATP + O2
Activity
1

7. 1- tube A. B. D. Signal (light)
2- A(control) No Signal
High Signal is (pieces tail)
Activity
1

8. Activity
2

9. Activity
2
H2O2 + indicator
molecule
(hydrogen donor )
Peroxidase
2H2O +
oxidized
indicator
molecules
(detectable
signal )

10. Prepare 3ml of a 1% peroxide solution .
Label 4 tubes (a.b.c.d)and follow the table
Prepare 0.1ml peroxidase darken the room .
Record all observations.
1
2
3
4
Activity
2

11. Test
Tube
Peroxide Indicator peroxidase Type of
signal
A 0.1ml 1%
H2O2
Tip-full of
luminol
peroxidase Light
blue
B 0.1ml 1%
H2O2
Tip-full of
sodium luminol
peroxidase Light
Faint
blue
C 0.1ml 1%
H2O2
0.2ml 4CN
solution .
peroxidase Purple
color
D 0.1ml 1%
H2O2
0.2ml TMB
solution .
peroxidase Yellow
color
Activity
2

12. Light . Blue signal and we use Tip-full of
luminal indicator .
Light . Faint blue signal . And we use Tip-
full of sodium luminal indicator .
Using 4CN solution as indicator . Give us
purple color signal .
We have yellow color signal . When we
use TMB solution as indicator .
Activity
2

13. Activity
3

14. Activity
3
Concentration of peroxide Observation of sensor strip
0.0003% 0cm
0.00075% 0.5cm
0.003% 1cm
0.0075% 2cm
0.025% 2.5cm
0.075 2.75cm
0.3 3cm
1% 3.5cm

15. 0.00%
0.20%
0.40%
0.60%
0.80%
1.00%
1.20%
Activity 3
observations of
sensor strip
Activity
3

16. The increasing of peroxide concentration , increase the
sensor strip observation .
The highest cone of peroxide = 1% Given us 3.5 cm
sensor strip .
Activity
3

17. Activity
4

18. Activity
4
Set up 6 tubes A-F
Set up a control (Tube A and 0.2ml 10% trition )
And the cholesterol sample
And the enzyme solution
1
2
3
4
And 2ml TMP indicator solution (to add)5

19. Activity
4
Test tube Cholesterol cone
1 0.5 F
1:2 0.25 E
1:4 0.125 D
1:8 0.0625 C
1:16 0.3125 B
0 0 A
A B
1:
16
C D
1:
4
E
1:
2
F
10
1:
8
Cholesterol +
O2
Cholesterol
oxidase
Oxidized form
of cholesterol +
H2O2

20. Activity
4
Tube (1-F) represent the high concentration of
cholesterol
Tube (B- represent 1:16 ratio) consider the lowest
concentration of cholesterol .

22. Enzyme
Glucose oxidase
+

23. H2O2 + indicator
molecule
(hydrogen donor )
Peroxidase
2H2O +
oxidized
indicator
molecules
(detectable
signal )

24. Tube Observation
2
(peroxidase)
Observation 1
(Glucose oxidase)
Result
A No color No color Water
B No color Blue Glucose
C Blue ------------ Peroxide
D No color Green Glucose

25. We put both enzymes and it does not
appear any reaction.
Give me Blue color when we put glucose
oxidase – (glucose)
Represent Blue color when we put
peroxidase – (peroxide)
We put glucose oxidase and give me
green color . (glucose)

27. Introduction.
Lactate is generated by lactate dehydrogenase
(LDH) under hypoxic or anaerobic conditions.
Monitoring lactate levels is, therefore, a good
indicator of the balance between tissue oxygen
demand and utilization and is useful when
studying cellular and animal physiology. D-
Lactate is produced in only minor quantities in
animals and measuring for D-lactate in
animal samples is a means to determine the
presence of bacterial infection.

28. samples

29. KIT CONTENTS

30. PROCEDURES
TubeA
TubeB
1- add 2.5m.l Buffer solution to A and B
Buffer solution Buffer solution
2.5m.l 2.5m.l

31. Tubestandard
2- add 3m.l Buffer solution to Tube standard .
Buffer solution
3m.l
PROCEDURES

32. 3- add 0.5m.l sample to tube A and B .
sample sample
PROCEDURES
TubeA
TubeB
0.5m.l 0.5m.l

33. 4- add 1 drop of enzyme A to Tube A
TubeAAssay buffer
EnzymeA
Add 1 drop
PROCEDURES

34. 5- add 1 drop of enzyme B to Tube B
Add 1 drop
NAD Solution
EnzymeB
TubeB
PROCEDURES

35. 6- Incubate for 60 min at room temperature .
59 00
PROCEDURES

36. 7- measuring by fluorescence spectator .
Read fluorescence λex/em = 530/585 nm
PROCEDURES

37. 6- measuring by spectator photometer
𝐷 − 𝐿𝑎𝑐𝑡𝑎𝑡𝑒 =
𝐹𝑠𝑎𝑚𝑝𝑙𝑒 – 𝐹𝑏𝑙𝑎𝑛𝑘
𝑆𝑙𝑜𝑝𝑒(µ𝑀 − 1)
× 𝑛 (µ𝑀)
Plot the D-lactate Standard Curve and
determine its slope. The D-lactate
concentration of the sample is computed
as follows:
where FSAMPLE and FBLANK are the fluorescence intensity values of the
Sample and Sample Blank respectively. Slope is the slope of the standard
curve and n is the dilution factor (e.g. n = 3 for serum samples).
If an internal standard was needed, the sample D-lactate concentration is
computed as follows:
𝐷 − 𝐿𝑎𝑐𝑡𝑎𝑡𝑒 =
𝐹𝑠𝑎𝑚𝑝𝑙𝑒 – 𝐹𝑏𝑙𝑎𝑛𝑘
𝐹𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 − 𝐹𝑠𝑎𝑚𝑝𝑙𝑒
× 27.8 (µ𝑀)

38. measuring by spectator photometer
where FSAMPLE and FBLANK are the fluorescence
intensity values of theSample and Sample Blank
respectively and FSTANDARD is the fluorescence
intensity value of the Sample plus Standard.
Note: if the sample ∆F value is higher than the ∆F
for 40 µM D-lactate standard or greater than the ∆F
for the internal standard, dilute the sample
in water and repeat the assay. Multiply the results
by the dilution factor.
𝐷 − 𝐿𝑎𝑐𝑡𝑎𝑡𝑒 =
𝐹𝑠𝑎𝑚𝑝𝑙𝑒 – 𝐹𝑏𝑙𝑎𝑛𝑘
𝐹𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 − 𝐹𝑠𝑎𝑚𝑝𝑙𝑒
× 27.8 (µ𝑀)

39. PROCEDURES
Summary
Pipetting (multi-channel) devices. Black, flat bottom 96-well plates and
fluorescent plate reader capable of reading at λex/em = 530/585 nm.
0
1
2
3
4
0 5 10 15 20 25 30 35 40
∆F×103
D-lactate

40. PROCEDURES
Recommendations

41. Thank all people, teachers, friends, and your school
that supported you during your journey in AL-bairaq
project.
Also don’t forget to thank Al-Bairaq team and the
sponsors who gave you the opportunity to participate
in AL-Bairaq project.