Still a draft! Kibak 2006

Determining the ATPase activity of each of your Mytilus gill tissue preparation fractions.

Outline:

• Measure Phosphate content of MgATP preparation.
• Measure Phosphate content of initial Mytilus homogenate.
• Combine MgATP + Mytilus fraction + buffer.
• Incubate 30 minutes.
• Measure Phosphate content after incubation using Malachite Green.
• Subtract initial phosphate and ATP phosphate from final phosphate to give “phosphate released” by ATPase activity.

Proceedure:

Assay Mixture is 2.5 mM ATP and  2.5 mM MgSO₄ in 0.8x RB.  You will receive a vial with about 9 mL.

Before starting assay, place 790 uL of assay mixture in tube #14 and add 10 uL dH2O.  This is a control.

Before starting assay, place 10 uL of crude mussel homogenate in tube #13 and add 790 uL RB.  This is a control.

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Prepare eight phosphate standard tubes numbered 0-7.  The first one contains only RB.

Place 800 uL RB in P_i Standard Tube #0 = 800 uL of 0 uM P_i 

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You will be given a tube containing Phosphate Standard Solution at a concentration of 40 uM P_i 

Place all your P_i Standard solution in Tube #1 = 1.5 mL of 40 uM P_i 

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Now remove 700 uL from Tube #1 and place in P_i Standard Tube #2

Add 700 uL RB to Tube #2 and mix well.  The concentration in Tube #2 is now 20 uM P_i 

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Now remove 600 uL from Tube #2 and place in P_i Standard Tube #3

Add 600 uL RB to Tube #3 and mix well.  The concentration in Tube #3 is now 10 uM P_i 

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Now remove 400 uL from Tube #3 and place in P_i Standard Tube #4

Add 600 uL RB to Tube #4 and mix well.  The concentration in Tube #4 is now 4 uM P_i 

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Now remove 200 uL from Tube #4 and place in P_i Standard Tube #5

Add 800 uL RB to Tube #5 and mix well.  The concentration in Tube #5 is now 0.8 uM P_i 

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Now remove 200 uL from Tube #5 and place in P_i Standard Tube #6

Add 800 uL RB to Tube #6 and mix well.  The concentration in Tube #6 is now 0.16 uM P_i 

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Now remove 200 uL from Tube #6 and place in P_i Standard Tube #7

Add 600 uL RB to Tube #7 and mix well.  The concentration in Tube #7 is now 40 nM P_i 

THESE ARE YOUR EIGHT PHOSPHATE STANDARDS TO USE IN PREPARING YOUR PHOSPHATE STANDARD CURVE

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Now you are ready to start the enzyme activity assay.

Place 790 uL of assay mixture in each of tubes 8-12.

At 1 minute intervals, add 10 uL of thawed and well-mixed sample to assay mixture and mix well again.

Final volume = 800 uL

Incubate 30 minutes before adding Malachite Green Reagent.

At T=30 minutes add 200 uL Malachite Green reagent to 800 uL of incubated assay mix plus sample.  Vortex tube and let incubate 10 to 20 minutes.

At T=40-50 minutes transfer to 1 mL cuvette and read absorbance at 650 nm.

TUBE #	PHOSPHATE	OD 650 nm
0	0 uM
1	40 uM
2	20 uM
3	10 uM
4	4 uM
5	0.8 uM
6	0.16 uM
7	0.040 uM
	SAMPLE
8	Mussel Tissue Homogenate
9	First Supernatant
10	First Pellet
11	Second Supernatant
12	Second Pellet
	CONTROLS
13	Mussel Tissue Homogenate w/o ATP
14	2.5 mM MgATP w/o sample