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

Buffer
(L) / PP-1
(L) / PNPP (30mM)
(L) / Sample
(L)
Blank / 50 / 10
Control / 40 / 10 / 10
Sample / 30 / 10 / 10 / 10

*Note: Do an assay with the stock MCLR as a control

Microcystin Standards

Concentration
(nm) / Volume of 100nM MCLR
(L) / Volume of H2O
(L)
0.5 / 5 / 995
1.0 / 10 / 990
2.0 / 20 / 980
3.0 / 30 / 970
4.0 / 40 / 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)
  4. collect load (in case it didn’t stick to the column)
  5. Wash with 10 mL 0.1% TFA/ 10% acetonitrile
  6. 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% beta-mercaptoethanol, 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)