Chloroplast Reduction of Indophenol

Reagents for reaction mix

apparatus set up

 

See previous protocol for the preparation of the purified chloroplasts. Continue to keep them ice cold until the moment you add them to the prepared tubes. Teams of two.

Supplies:
purified spinach chloroplasts, ice cold
0.1 M PO4 buffer, pH 6.5 (pH 7.0 OK?)
2.5 x 10-4 M 2,6 dichlorophenolindophenol
(36.3 mg DCIP/500 mL, A600 = 3.0)
Equipment/ team
10 uL and 200 uL displacement pipets with tips
seven cuvettes (or unblemished 13×100 mm test tubes)
100 watt light
meter stick
37C incubator (can exclude light, for whole class)

apparatus set up

Set up apparatus:
a: 37C hot block (for 13×100 mm tubes) nearby and warmed up.
b: 37C incubator warmed up.
c: spectrophotometer on the same desk as the light exposure apparatus.
d: 100 watt bulb with reflector 25 cm from open test tube rack.

Construct a data table in your notebook to accept time course data (See below).

Select and label seven cuvettes (or six very clean, unblemished 13 x 100 mm test tubes to serve as cuvettes) (B for blank + six tubes).

Reagents for reaction mix

Prepare DCIP reaction mix which should have an A600 of 0.400 to 0.600.
Per team of two:
12 mL 0.1 M PO4, pH 6.5 buffer
12 mL 0.5 M sucrose
8 mL 2.5 x 10-4 M DCIP

Dispense 4 mL of the reaction mix to each of your tubes with repeater pipetter.

5. Add all of the following ingredients except for the chloroplasts:

Table for recording DCIP reduction A600 versus time of light exposure.

Table for recording DCIP reduction A600 versus time of light exposure.

Copy this table into your notebook prior to starting the chloroplast reduction experiment. Every blank square gets a reading.

6. Prewarm prepared tubes (lacking chloroplasts) in 37C hot block for 2 min.
7. Add diluted chloroplasts to tubes 3 and 5. Mix and read the A600 of 1, 3, and 5 against a water blank (= T0 for dark tubes). Place immediately in a 37C incubator, keep light excluded.
8. Read A600 of tube 2, place in front of the light
9. Add 10 uL of chloroplasts to tube 4, mix well, read A600, place in front of the light, and start the stopwatch.
10. Add 20 uL of chloroplasts to tube 6, read A600, place in front of the light, when the stopwatch reads 30 seconds.
11. At 30 second intervals, alternatively read the A600 of 4 and of 6 for 10 minutes. At 5 minutes, also read tube 2. Read tube 2 again at 10 min. (Keep tubes clean, read in consistent configuration.)
10. At 15 minutes, read A600 of all tubes, including those which were kept in the dark.
11. Plot the absorbency of each tube versus time.
•Discuss the significance of the differences between the various six curves.

Here is a graph of reduction of DCIP by 20 uL of chloroplast suspension

Isolation of Chloroplasts by Differential Centrifugation

grind spinach with sand as a grinding aid

decant after first centrifugation

Equipment and Supplies per team of two students:

SUPPLIES
Fresh spinach
clean sharp sand
50 mL 0.5 M sucrose (17% w/v)
cheese cloth, 12 x 12 inches
ice

EQUIPMENT
Styrofoam cooler for ice bath
25 mL graduated cylinder
mortar and pestle (or blender)
gtable top clinical centrifuge
glass filter funnel
two 16×150 mm test tubes in rack
three 13×100 mm test tubes in rack
plastic capped 15 mL centrifuge tube
double pan balance
glass stirring rods
KEEP ALL EQUIPMENT AND MATERIALS ICE COLD:
Per pairs of students:

grind spinach with sand as a grinding aid

Prepare, weigh and homogenize:
Grind 8 g deveined spinach with ½ tsp clean sharp sand in mortar and pestle to a paste.

Suspend in 0.5 M sucrose:
Measure out 16 mL ice-cold 0.5 M sucrose solution in a 25 mL graduated cylinder. Add in 3-4 mL increments, grind to smooth pulp with each addition. (A blender may be used for >100 mL volumes)

Filter
homogenate through about eight layers of clean cheese cloth in a glass funnel into an iced 16×150 mm test tube.

Pour filtrate back into 25 mL cylinder and record volume. Save ~0.5 mL of the filtrate (F1) in a labeled 13×100 mm test tube to examine at 400x under microscope to determine composition and illustrate in notebook. Note appearance of components and degree of heterogeneity. (Label cells, ghosts, chloroplasts, mitochrondria, debris.)

Centrifuge at low speed: prepare a balance tube against the filtrate in a 16×150 tube and spin at 50x g for 10 minutes (speed 2 on the clinical centrifuge).

decant after first centrifugation

Decant the top 10 mL into a clean cold centrifuge tube, discard sediment. Record volume. Save ~0.5 mL supernatant (S1) to examine under microscope to determine composition, illustrate and label as in step 2.

Centrifuge the supernatant from step 3 opposite a carefully balance tube at 1000x g for 10 minutes (speed 7) to precipitate chloroplasts. How does the supernatant appear? Precipitate? Carefully decant all of the supernatant into 16×150 mm tube but save the pellet. Discard supernatant if you have a significant pellet. (You will lose some soft pellet, but not to worry.)

Resuspend pellet from step 4 to 1/10th of the volume of the step 2 filtrate in ice-cold 0.5 M sucrose with a clean, ice cold stirring rod. Record final volume. Keep on ice at all times. Examine suspended organelles (SO) under microscope to determine composition, illustrate as is step 2. [You should have illustrations of F1, S1 and SO in your book.]

Features of Major Joints

shoulder, superior view

knee, extended posterior view

Examine the models provided and identify the following features of these joints. Articulated joints will be brought in from a local butcher, and you should identify these features on them and illustrate the specimen. Note that wordstems will help you tremendously in locating and understanding the connections these features make.menisci

Here are pictures taken of a dissected knee and hip from a deer (thanks to Kathy and Andy):
Here are images of recently purchased joint models.

ILLUSTRATE SYNOVIAL JOINT FEATURES:

These are common to all synovial joints: (Martini’s 6th, p 266)
synovial capsule A collagenous structure which encloses, supports and protects the joint.
It often incorporates ligaments into its walls.
synovial membrane Forms the inner lining of capsule, secretes synovial fluid
articular cartilage Hyaline cartilage padding on the articulating surfaces of joined bones
synovial fluid A lubricating, nourishing fluid rich in mucopolysaccharide. Contained within the capsule.

Illustrate each joint from the perspective specified, label features you can see, not the ones in brackets which cannot be seen:

superior view of shoulder:

shoulder, superior view

Here is a labeled version SHOULDER (glenohumeral): (lateral view) (Martini’s 6th, p 277)
clavicle, scapula, humerus glenohumeral ligament (divided into three parts, superior, middle (towards the froknt) and inferior (split in two) portions)
[coracohumeral ligament (not on model) ] [glenoid labrum (hidden on model) ]
transverse humeral ligament (bridges the greater and lessor tubercles)
tendon of long head biceps brachii (lies in the intertubercular groove)
tendon sheath of long head of biceps brachii [musculotendinous cuff (not on model)]
Here is a labeled version

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ELBOW: (superior, anterior view) (Martini’s 6th, p 278)
humerus, ulna, radius radial collateral ligament (or lateral) lateral epicondyle
annular ligament and radia notch on ulna
ulnar collateral ligament (or medial)
medial epicondyle
anterior ligament [coronoid process , under the anterior ligament] [tendon of triceps (not on model, would be attached to olecranon process)]
Here is a labeled version

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lateral view

medial view

posterior view

KNEE JOINT: (lateral view) (Martini’s 6th, pp 281).
femur, patella, tibia, fibula lateral collateral ligament [medial collateral ligament, seen in medial view]
anterior cruciate ligament: indicate with dotted lines (the lateral of the two cruciate ligaments)
posterior cruciate ligament : indicate with dotted lines
popliteal ligaments (multiple, at the rear of the knee)
patellar ligament (attaches the patella to the tibial tuberosity, famous for it use in the patellar reflex text)
lateral meniscus [medial meniscus, seen in medial view]

Here is a labeled view of the proximal tibial features of the knee joint from a deer

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flexed knee

extended knee

Knee, showing the way that the:
anterior cruciate ligament tightens upon extension of the leg
posterior cruciate ligament prevents femur from sliding forward on the tibial surface, especially during flexion of the leg.
Another model of the flexed knee.
Here are the cruciate ligaments of a chicken knee joint, labeled.
Here is a labeled version, extended
Here is a labeled version flexed

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HIP JOINT: Illustrate two views of the hip, anterior and posterior.

Here is the dissected hip joint labeled.

HIP JOINT ANTERIOR:
(Martini’s 6th , p 279)
ilium, pubis, ischium, femur capsule strengthened by these ligaments:
anteriorly
iliofemoral ligament (numbered #3 on the model)
pubofemoral (or pubocapsular ) ligament (numbered #4)
Another model of the anterior hip.

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HIP JOINT, POSTERIOR: (Martini’s 6th , p 279)
posterior view, capsule strengthened by these ligaments:
ischiofemoral (or ischiocapsular ) ligament (numbered #5) iliofemoral ligament (at the top)
[ligamentum teres ties femur to os coxa, hidden by ligaments]
[acetabular labrum lip of cartilage, hidden by ligaments ]
Another model of the posterior hip.

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Medial view of the os coxa with a portion cut out to show the head of the femur and the ligamentum teres (numbered #6). It also shows the obturator ligament

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Illustrate two views of the hip, anterior and posterior:

Protein Assay by Microbiuret: Standardization

From DBF’s Hopkins Notebooks, III, p. 102 & VI, p. 75.

Adding microbiuret

tubes ready to read

Microbiuret reagent is an alkaline solution of copper ions which complex with the peptide bonds in protein to produce a blue-purple color (absorption max = 310 nm) . Following Beer’s Law, the color intensity of a small amount of protein mixed with this reagent should be proportional to protein concentration. Thus, this reagent can be used to assay soluble protein in unknown solutions. To do this, a “standard curve ” must be generated by assaying known amounts of protein . That is the purpose of this first exercise in this series.

Wear safety goggles and handle the microbiuret reagent with care since it is a dangerous caustic solution of 35% NaOH (lye). Any hint of slipperiness on your fingers should be rinsed off with a solution of 5% boric acid (in squirt bottles) followed by thorough hand washing.

FOR A TABLE OF TWO STUDENTS (perform experiment in pairs):

EQUIPMENT
3 test tube racks: 2 for 13×100 mm, 1 for 16×150 tubes
12 13 x 100 mm test tubes
2 16×150 test tubes
1 5 mL pipet for water (in 16×150 test tube)
1 2 mL pipet for protein solution (in 16×150 test tube)
pipetman pipet bulb
vortex
safety glasses
spectrophotometer, warmed up
two cuvettes in test tube rack

SUPPLIES
8 mL microbiuret reagent 2 in Eppendorf pipettor or in 1 mL repipet
(front of room)
30-40 mL dH 2O in 125 mL flask
mL of mg/mL protein standard 3 in 13×100 test tube
Kimwipes, paper towel

STANDARDIZATION OF MICROBIURET REAGENT:
1)  Write out an experiment table in your book  (see Sample Layout of an Experiment)
2)  Then set up 13×100 mm tubes for the standardization
3)  Then add in sequence, accoding the to volumes in the following table

Here is the table for this standardization:

a) add water first (Always add the least expensive reagents first unless there is a compelling reason to do otherwise)

b) add the appropriate volume of protein.

Adding microbiuret

c) add microbiuret reagent using a repeating pipetter.
Here is a picture illustrating the use of the Beckman Repeater

Here are the steps illustrated for setting up the standardization:

4)  Vortex to mix well, let sit 15 min
5)  Read tubes: use tube B as the blank, and read A₃₂₅ in a spectrophotometer. Read at 310 nm if you have a UV capabilities.
6)  Calculate the mg protein (or in each tube. (1 g = 10 ³ mg = 10 ⁶ ug).

microbiuret standardization curve

microbiuret standardization data

7)  Plot standardization curve (protein vs A ₃₂₅) .  Here is a typical standardization curve.
8)  Determine conversion factor to convert from optical density at A ₃₂₅ (OD) to mg protein: Determine the slope of the line where the curve is linear.  The slope will be approximately 1.25 mg/OD unit at A ₃₂₅ ).

Wash work areas well when finished to clean up any spilled caustic materials.

¹ Our Spectronic 20s cannot measure in the UV range, but only measure absorbency down to 325 nm.

² MICROBIURET REAGENT: (Safety glasses should be worn during this experiment since this solution is close to a 20% solution of NaOH. Handle with extreme caution.)

Solution A:
40 g NaOH (caution, caustic)
100 mL dH2O to dissolve NaOH with caution

Solution B:
400 mg CuSO4
40 mL water, agitate to dissolve
1) Q.s. To 150 mL with dH2O
2) Add solution B slowly to solution A with stirring.
Store in labeled bottle marked CAUTION : caustic

3 STANDARD PROTEIN, mg/mL: Prepare 1 mg/mL solution of standard protein (bovine serum albumin [BSA], or egg albumin) by adding 100.0 mg of powder to 80 mL dH₂ O, thoroughly dissolve with stirring, avoiding foaming which denatures protein. If possible, let sit at 4C for a week to completely dissolve. Q.s. to 100.0 mL. Store at 4C. (Need ~7 mL/student).

For determination of  protein in unknowns, see:

SAMPLE LAYOUT OF AN EXPERIMENT: