PNPP Inhibition Assay
An, J-S., Carmicheal, W.W. (1994) Toxicon 32, 1495-1507
Buffer
50mM Tris
0.1 mM EDTA
30 mM MgCl2( or MgOAc)
-adjust pH to 8.3
1mg/mL BSA
Before use:
0.5 mM MnCl2
0.2 % -mercaptoethanol (BME)
Assay:
volume = 60L
Absorbance wavelength = 405nm
Blank
Control
Sample
Buffer
(L)
50
40
30
PP-1
(L)
10
10
PNPP (30mM)
(L)
10
10
10
Sample
(L)
10
*Note: Do an assay with the stock MCLR as a control
Microcystin Standards
Concentration
(nm)
0.5
1.0
2.0
3.0
4.0
Volume of 100nM MCLR
(L)
5
10
20
30
40
Volume of H2O
(L)
995
990
980
970
960
HPLC
All HPLC runs were on a Beckman with a microbore Vydac C18 column. HPLC columns
were equilibrated with 0.1% TFA/water and eluted with linear AB gradient
(0.3%B/minute), where solvent A is 0.1% TFA/water and solvent B is 0.1%
TFA/acetonitrile, over 3 hours at a flow rate of 0.2 mL/minute. The wavelength for
detection was 206nm.
Reaction: Microcystin-LR and aminoethanethiol (cysteamine)
Moorhead, G., MacKintosh, R.W., Morrice, N., Gallagher, T., MacKintosh, C. (1994)
FEBS Letters 356, 46-50
Carry out in Fumehood:
For 1mg of MCLR:
Use “cow” to purge all solvents with N2 gas
-3mL H2O
-2mL DMSO
-1mL 5N NaOH
Prepare 1mg/mL aminoethanethiol (found in fridge, REALLY STINKY! hard to
dissolve, will need to vortex)
Reaction Vessel (tube)
-1.5 mL H2O
- 2 mL DMSO
-0.67 mL 5N NaOH
-1g aminoethanethiol-HCl
-1mL of pooled MCLR 78-84B (~0.9mg/mL)
-vortex to mix (STINKY!!!)
-purge under N2 gas at 52oC for 30 minutes
-added 6.5 mL glacial acetic acid
-added ~50 mL 0.1% TFA/ H2O
-pH to 1.5 with 100% TFA
Purification with Sep Pak
See Sep-Pak Manual p.15 Elution Protocols: Reverse Phase Chromatography
1. Solvate with 10 mL 0.1% TFA/acetonitrile
2. Flush with 10 mL 0.1% TFA/water
3. Load sample (~60 mL in total)
a. collect load (in case it didn’t stick to the column)
4. Wash with 10 mL 0.1% TFA/ 10% acetonitrile
5. Elute with 0.1% TFA/ acetonitrile until you don’t see anything with PNPP
inhibition assay (in other words
-dry elutions with Speed Vac
Reaction: Aminoethanethiol-MCLR and NHS-activated Sepharose
Moorhead, G., MacKintosh, R.W., Morrice, N., Gallagher, T., MacKintosh, C. (1994)
FEBS Letters 356, 46-50
See Manual
Buffers:
50mM NaHCO3 pH 9.2
50mM Tris-HCl pH 8
0.5M NaCl
0.5M NaCl, 50mM NaOAc pH 4
Coupling-Ligand Solution
Dissolve Aminoethanethiol-MCLR in 1.0-2.5 mL 50mM NaHCO3 Buffer (in as little
volume as possible, my solution was cloudy but I went ahead with the experiment
anyways)
Removal of Isopropanol
-shake bottle and take 6 mL
-centrifuge (2500rpm-3000rpm for 5 minutes)
-take off supernatant
-wash with 10-15 medium volumes of cold 1mM HCl
-remove
Coupling
-add ligand solution
-pH to pH 8-9
-incubate end over end for 3-4 hours at room temperature or overnight at 4oC
Blocking
-centrifuge
-remove supernatant, keep aliquot for HPLC
-add 50mM Tris-HCl pH 8 for blocking (added up to 10mL total resin and solution)
-incubate end over end overnight at 4oC
Washing
for each wash, add, mix, centrifuge, remove
-alternately wash with 50mM Tris pH 8, 0.5M NaCl 50mM NaOAc, and 0.5M NaCl
-repeat 5 times
-store in 20% ethanol
Control
(different Sepharose than that used for coupling with MCLR)
See manual
-measure mass and add 50mM (or high concentration) Tris-HCl pH 8
-incubate end over end overnight at 4oC
Binding Experiments
-this I made up myself
Buffers:
“Buffer A” = 50mM Tris-HCl, 0,1mM EDTA, 0.5mM MnCl2, 0.2% betamercaptoethanol, pH 7.5
Buffer A with 0.3M NaCl
Buffer A with 3M NaSCN
PP1 Solution
-I made up solutions of 0.075mg/mL (dilute PP1 with Buffer A)
-when I made the solutions too concentrated I seemed to get less efficient binding, maybe
the salt concentration was too high? I don’t know. You can play around with it maybe
you’ll have better luck than me
Resins
-I used 50mL of resin (just calculate based on the total volume of the slurry and the
volume of resin)
-remove the 20% ethanol, wash with water then buffer or with buffer (Marcia mentioned
that salts might precipitate in ethanol?)
-add the PP1 and incubate for 1 hour
-wash with 0.3M NaCl Buffer
-elute with 3M NaCl
for PP2A
Buffer A with okadiac acid (large excess)