3.

MEMBRANE PREPARATION

3.1. RAT BRAIN MEMBRANE PREPARATION FOR CTX AND STX RBA

Apparatus
- Teflon/glass homogenizer (i.e., motorized Teflon pestle and glass tube);
- Motorized tissue homogenizer (Polytron or small handheld blender);
- High speed centrifuge and fixed angle rotor capable of 20,000 x g (rcf);
- Centrifuge tubes (12–15 mL) rated for > 20,000 x g (rcf);
- 2 mL plastic cryovials;
- 300 or 500 mL graduated beaker;
- Disposable 5 and 10 mL pipettes;
- Forceps for handling rat brains.

Reagents
- 20 rat brains, male Sprague-Dawley, six wk old (Hilltop Lab Animals);
- 1L buffer pH 7.4: 100 mM MOPS, pH 7.4 (Sigma, St. Louis) containing 100 mM choline
chloride (Sigma), 0.1 mM PMSF*
*PMSF (phenyl methylsulfonyl fluoride, Sigma) stock preparation (0.1 M): dissolve
0.0174 g PMSF in 1 mL isopropanol; a larger volume may be prepared and stored in 1
mL aliquots at -20oC (e.g. 0.174 g in 10 mL isopropanol). Prior to use thaw PMSF and
thoroughly mix to redissolve completely; then add to 1L buffer fresh daily at each use.
Do not refreeze aliquots.
- Protein Assay Reagent Kit: the protein concentration of the membrane preparation is
determined using the Micro BCA (Pierce Micro BCA Protein Assay Reagent Kit #23235,
microplate method) or #23225 (tube method) protein assay kit or equivalent protein assay.

17
Procedure

1. Remove medulla from each brain using ‘pointed’ forceps and discard. 1
2. Place cerebral cortices in ice-cold buffer pH 7.4 and place container on ice.
Homogenize two brains in 25 mL buffer pH 7.4 (12.5 mL per brain) in a glass
homogenizer tube with a Teflon probe. Homogenize with 10 up and down
strokes at 385 rpm on ice slurry. Keep tube in ice at all times.
Pour homogenized tissue into 250 mL beaker on ice and repeat procedure with
remaining cortices

2
3. Pool homogenized tissue and transfer to high speed centrifuge tubes.
Centrifuge at 20,000 x g for 15 minutes at 4°C.
Quickly aspirate (or pour off) supernatant and resuspend pellets in 10 mL
buffer pH 7.4 per brain (200 mL for 20 brains).
3

4. Pool resuspended membrane preparation in a 500 mL beaker and keep on ice.

Polytron at 70% full speed for 20 seconds.

5. Aliquot 2 mL/tube in cryogenic vials: cryotube must be kept on ice and mixed
prior each aliquot.
Freeze and store at -80°C. This preparation is stable for at least 6months.
4 5

6. Determine protein concentration of the membrane preparation and hence the

dilution needed for a final 1mg/mL protein concentration in the assay

18
3.2. SF9 MEMBRANE PREPARATION FOR DA RBA

Apparatus
- High speed centrifuge and fixed angle rotor capable of spinning at 21,000 RCF (x g) at 4 °C.
- 500 W sonicator with microprobe
- High speed centrifuge tubes (12–15 mL capacity) capable of withstanding speeds up to 21,000
RCF (x g)
- 2 mL plastic cryovials
- disposable pipettes for buffer handling
- Analytical balance to ensure equal weight of tubes at high speed

Reagents
- Cell pellet from transfected sf9 cells provided by TexCell (pellet equivalent to 200 mL of
cells). This is kept at -80°C until thawed.
- Ice
- 1L Tris buffer buffer pH 7.1 with 0.1mM PMSF

*PMSF (phenyl methylsulfonyl fluoride, Sigma) stock preparation (0.1 M): dissolve 0.0174 g
PMSF in 1 mL isopropanol; a larger volume may be prepared and stored in 1 mL aliquots
at -20oC (e.g. 0.174 g in 10 mL isopropanol). Prior to use thaw PMSF and thoroughly mix to
redissolve completely; then add to 1L buffer fresh daily at each use. Do not refreeze aliquots.

Procedure
1. Precool high speed centrifuge to 4°C. Set speed to 21,000 RCF (xg) and insert fixed angle rotor.
2. Remove 15 mL tube of cells from -80˚ freezer and thaw on ice. Assumption is that these cells are
equal to 200 mL Sf9 cells.
3. After cells are completely thawed, sonicate in ice bath using probe sonicator at setting 6 for short
bursts adding up to 30 seconds.
4. Distribute sonicated cells evenly into 8 high speed centrifuge tubes and fill to the top with 50mM
Tris buffer (pH 7.1) containing 0.1mM PMSF. It is important to fill tubes to the top and evenly
because if not, they will collapse during centrifuging. Check weights of tubes on a balance.
5. Spin tubes for 20 minutes at 4˚C at 21,000 RCF.
6. Retrieve tubes from centrifuge and decant supernatant into sink. Add 1mL Tris to each tube and
vortex to pop pellets off the bottom of the tube. Combine pellets into one tube and break pellets
into very small pieces using a small stirring rod to homogenize distribution.
7. Redistribute the 8 mL of prep into 8 new centrifuge tubes and fill each tube with Tris buffer as in
step 4.
8. Repeat steps 5–7 two more times, for a total of three spins in the ultracentrifuge. Be sure to
balance tubes in the centrifuge.
9. After the third spin, combine and mix pellets as in step 6, then resuspend prep to 20 mL in a 50
mL centrifuge tube.
10. Sonicate on ice bath in bursts adding up to 30 seconds at setting 6.
11. Prepare 20 1 mL aliquots in 2 mL Nalgene cryovials. Label with correct batch number and date.
Store at -80˚C for future use. Note: This is a suspension, not a solution, so mixing is essential
when making aliquots.

19
 4. RBA FOR PARALYTIC SHELLFISH POISONING TOXINS: SAXITOXINS

4.1. APPARATUS, SUPPLIES AND REAGENTS

Apparatus and Supplies

- Microplate scintillation counter
- Micropipettors (1–1000 L variable volumes) and disposable tips
- 8 channel pipettor (5–200L variable volume) and disposable tips
- 96 well microtiter filter plate with FB glass fiber filter/ 0.65 m pore size
- Duropore support membrane (Millipore, Bedford)
- Multiscreen vacuum manifold (Millipore)
- Vacuum pump or house vacuum
- 15 and 50 mL conical plastic centrifuge tubes
- Mini-dilution tubes in 96-tube array
- Solvent reservoirs
- Ice bucket and ice
- Vortex mixer
- Plate sealing tape (Millipore)
- 1L volumetric flask
- -80°c freezer
- -20°c freezer
- Refrigerator
- pH meter or pH paper
- Hot plate
- 10 mL graduated cylinder
- Centrifuge for 15 mL tubes
Reagents
- 0.1 and 1 M hydrochloric acid (HCl)
- 0.1 M NaOH
- Water, deionized (dH2O; 18 µOhm)
- [3H] STX (0.1 mci/mL, 24 Ci/mmol, >90% radiochemical purity; ARC)
- STX diHCl reference standard (NIST RM 8642 (www.nist.gov).
- 100 mm MOPS, pH 7.4, 100 mm choline chloride
- Rat brain membrane preparation
- Liquid Scintillant: Optiphase liquid scintillation cocktail (Perkin-Elmer Life Sciences)

20
4.2. SAMPLE PREPARATION

4.2.1. STX extraction from shellfish

1. Shuck shellfish to obtain ~ 100 g of meat.

Drain on a sieve and homogenize 3 minute in a blender Homogenate may be
stored frozen at -20oC
1

Drained shellfish (left) and blender (right)

2. Accurately weigh 100 g tissue homogenate into a 2 3

tared 500 mL beaker.
3. Add 100 mL of 0.1 N HCl, stir thoroughly and
check pH (Fig.10 Right). The pH should be < 4.0,
preferably 3.0. If necessary, adjust pH as described
below.

Weigh (left) and resuspend in 0.1N HCl (right)

4. Heat mixture, boil gently for 5 minutes, and cool to room temperature.
Adjust cooled mixture to pH 3.0-4.0 (never > 4.5) as detected by a pH meter or pH paper.
To lower pH, add 5 N HCl dropwise with stirring; to raise pH, add 0.1 N NaOH dropwise 5
with mixing to prevent local alkalinization and consequent destruction of toxin.
5. Transfer mixture to graduated cylinder and dilute to 200 mL.
4

6. Return mixture to beaker, stir to homogeneity, and let settle until a 6

portion of the supernatant is translucent.
7. Pour approximately 15 mL of the supernatant into a centrifuge tube.
Centrifuge at 3,000 x g or higher for 10 minutes.
8. Filter supernatant through 0.45 µm nylon filter (may need GF/F
8
glass fiber pre-filter).
Store extracts at -20°C until tested with receptor binding assay

Centrifuge (top) and filter (bottom)

21
 4.2.2. STX extraction from phytoplankton

1. Tare dry grinding/extraction tube, place filter 1

at bottom, record weight.
Add desired amount of 0.1 N HCl to tube,
record weight and mix.
Note: Use 2.5 mL 0.1 N HCl for 25 mm
filters, 5 mL for 47 mm filters

Tare (left) and place filter (right)

2. Break up filter by grinding with Teflon 2

pestle at 250 rpm for 1 min (2 min for 47
mm filters). Wash pestle with 0.1 N HCl
between samples.
Centrifuge for 1 min at 45 x g.
Rinse upper edges of tube with supernatant.

Teflon pestle

3. Sonicate on ice slurry for 1 min for 25 mm 3

filters (2 min for 47 mm filters) using 500 W
sonic disrupter with a microprobe at the
maximum allowable setting. Rinse
microprobe with 0.1 N HCl between
samples.

Probe sonicator in soundproof box (left)

microprobe in tube (right)
4. Heat tubes to 100°C in a boiling water bath 4
for 7 min.
Transfer sonicated and boiled sample to a 15
mL conical tube and centrifuge for 5 min at
700 x g to pellet filter particles.
Remove supernatant using a 3 or 10 cc
syringe and pass through a 0.22 µm filter
into a 2 or 5 mL cryogenic vial.
Store samples at -20°C until analysis.

Water bath

22
4.3. RECEPTOR BINDING ASSAY PROCEDURE

4.3.1. Preparation of stock solutions and standards for assay

Assay Buffer MOPS (100nM) choline chloride (100nM), pH 7.4

- weigh out 20.9 g of MOPS (3-morpholinopropanesulfonic acid) and 13.96 g of choline
chloride and add to 900 mL dH20
- adjust pH to 7.4 with NaOH while stirring
- transfer quantitatively to volumetric flask and bring to a final volume of 1 L with dH20
- store at 4°C

Radioligand solution: [3H]STX

Stock [3H]STX is provided in 50 µCi ampoules, 24 Ci/mmol, 0.1 mCi/mL (equivalent to 4.17 μM).

- Prepare a 15 nM working solution of [3H]STX for each assay fresh daily:

14 µL of stock [3H] STX + 3.86 mL Assay Buffer (this will provide a 2.5 nM final
concentration in microplate wells). Note that the amount of [3H] STX added may vary
according to the specific activity of the stock [3H]STX.
- Measure total counts of each working solution prior to running an assay: add 35 µL of the
[3H]STX working solution to a liquid scintillation vial with 4 mL scintillant and count using a
liquid scintillation counter. A volume of 35 µL of the [3H]STX working solution should
contain 27,650 dpm. Depending on the efficiency of the scintillation counter used, the
corresponding CPM will vary, but should be consistent day to day and within 15% of the
expected value.

Unlabelled STX reference standard

STX diHCl reference standard NIST RM 8642 is provided at a concentration of 268.8 µM
(100µg/mL).
A “bulk” serial dilution for the standard curve is made up in advance and stored at 4ºC for up to 1
month. The use of bulk reference dilutions minimizes the pipetting needed for setting up an assay
routinely and improves day to day repeatability.

To make up the standard curve perform the following STX diHCl serial dilutions in 0.003 M HCl
(from a 3 M stock, 50 µL in 50 mL)
Stock In assay*
100 µL 268.8 µM STX + 4.38 mL 0.003 M HCl 6x10-6 M 1x10-6 M
500 µL 6x10-6 M + 4.5 mL 0.003 M HCl 6x10-7 M 1x10-7 M
-7 -7
1.5 mL 6x10 M + 3.5 mL 0.003 M HCl 1.8x10 M 3x10-8 M
-7 -8
500 µL 6x10 M + 4.5 mL 0.003 M HCl 6x10 M 1x10-8 M
500 µL 1.8x10-7 M + 4.5 mL 0.003 M HCl 1.8x10-8 M 3x10-9 M
-8 -9
500 µL 6x10 M + 4.5 mL 0.003 M HCl 6x10 M 1x10-9 M
-9 -10
500 µL 6x10 M + 4.5 mL 0.003 M HCl 6x10 M 1x10-10 M
5 mL 0.003 M HCl 0M Reference

*All standards are diluted 1/6 in the assay.

Unknown samples
Perform two initial dilutions at 1:10, 1:50 and 1:200 in 0.03M HCl of unknown samples (extracted and
stored in 0.1N HCl) of using dilution tube (Fig. 9.)

Fig. 9. Dilution tubes

23
Inter-assay calibration standard (QC check):
A reference standard containing 1.8 x 10-8 M STX (3.0 x 10-9 M STX in assay) is prepared in advance
in 0.003 M hydrochloric acid and kept frozen (-80ºC) in 0.5 mL to 1 mL aliquots for long term
storage. Aliquots should be thawed and stored at 4ºC for routine use (stable up to1 month) and
included undiluted (35 µL) in each analysis (triplicate wells). This serves as a QC check and confirms
day-to-day performance of the assay.

Rat brain membrane preparation:

 Add 2mL thawed rat membrane preparation to 18 mL MOPS, vortex
 Pour diluted prep into empty solvent reservoir

4.3.2. Performing the assay

4.3.2.1. Plate setup and incubation

When possible, use a multichannel pipette to minimize pipetting effort and increase consistency
(Fig.10.). Standard curve, QC check, and sample extracts are run in triplicate. Multiple dilutions of
sample extracts need to be analyzed in order to obtain a value that falls on the linear part of the
competition curve for quantification.

FIG.10. Plate setup using a multichannel pipette

For ease of analysis, it is convenient to use a plate layout which maximizes the number of samples and
reference standards that can be analyzed on one plate (a layout example is provided in Fig. 11.) For
shellfish extracts, a minimum dilution of 1:10 is used, which minimizes potential matrix effects while
still providing a limit of quantification of approximately 30 g/kg shellfish:

1 2 3 4 5 6 7 8 9 10 11 12
-6 -6 -6
A 10 10 10 QC QC QC U3 U3 U3 U6 U6 U6
1:50 1:50 1:50 1:10 1:10 1:10
B 10-7 10-7 10-7 U1 U1 U1 U3 U3 U3 U6 U6 U6
1:10 1:10 1:10 1:200 1:200 1:200 1:50 1:50 1:50
C 3x 3x 3x U1 U1 U1 U4 U4 U4 U6 U6 U6
10-8 10-8 10-8 1:50 1:50 1:50 1:10 1:10 1:10 1:200 1:200 1:200
D 10-8 10-8 10-8 U1 U1 U1 U4 U4 U4 U7 U7 U7
1:200 1:200 1:200 1:50 1:50 1:50 1:10 1:10 1:10
E 3x 3x 3x U2 U2 U2 U4 U4 U U7 U7 U7
10-9 10-9 10-9 1:10 1:10 1:10 1:200 1:200 1:200 1:50 1:50 1:50
F 10 -9 10 -9 10 -9 U2 U2 U2 U5 U5 U5 U7 U7 U7
1:50 1:50 1:50 1:10 1:10 1:10 1:200 1:200 1:200
G 10 -10 10 -10 10-10 U2 U2 U2 U5 U5 U5
1:200 1:200 1:200 1:50 1:50 1:50
H REF REF REF U3 U3 U3 U5 U5 U5
1:10 1:10 1:10 1:200 1:200 1:200
U = unknown sample
Fig.11. Typical 96 well plate layout

24
 Add in the following order to each of the 96 wells:
35 μL assay buffer (assay buffer is added first in order to wet the filter membrane)
35 μL STX standard, QC check, or sample extract
35 μL [3H] STX
105 μL membrane preparation
 Cover and incubate plate at 4oC for 1 hour.

4.3.2.2. Assay Filtration and Counting

Assay Filtration 1
1. Place 96-well plate on the MultiScreen vacuum manifold. Fill empty
wells with 200 µL of assay buffer to ensure even filtration across plate
2. Turn on vacuum. Optimum vacuum will pull the wells to dryness in 2-
5 sec. Pull contents of all wells through until liquid is removed.
3. With vacuum still running, quickly rinse each well twice with 200 μL
ice cold assay buffer using multichannel pipette

Counting
4. Remove the plastic bottom from the plate. Gently blot the bottom once on
absorbent toweling
5. Place microplate in a counting cassette. Seal the bottom of the 96-well
plate with sealing tape
6. Add 50 μL scintillation cocktail per well using multichannel pipette
7. Seal top of plate with sealing tape.
8. Allow to sit a minimum of 1 hour at room temperature
9. Tag the plate
10.Count using a microplate scintillation counter

4 5 6 7

8 9 10

25
4.3.3. Analysis of data

Sample quantification is carried out only on dilutions that fall on the linear part of the competition
curve (Bi/B0 = 0.2-0.7). Principles and assay Quality Control acceptance are detailed in the chapter
2.2.3 Analysis of Data, Page 15 of this manual.
Where more than one dilution falls on the linear part of the curve, all sample wells corresponding to
these dilutions are used to calculate sample concentration. Sample concentration is calculated in μg
STX equivalents/kg shellfish, using the following formula:

(nM STX equiv) x (sample dilution) x (210 μL total volume) = nM STX equiv in extract
35 μL sample

(nM STX equiv in extract) x 1L x 372 ng x 1 μg = μg STX equiv/mL

1000 mL nmol 1000 ng

μg STX equiv/mL x mL extract x 1000g = μg STX equiv/ kg

g shellfish extracted kg

26