Gabor Hormone
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PROTOCOL FOR STEROID EXTRACTION FROM WATER
Always wear gloves
If necessary, filter all samples prior to extraction- Q8 filter paper
Never allow the pressure in the manifold to exceed 20 mmHg
Clean all beakers, tubes, vials, etc. with 100% ethanol and DI water to avoid
contamination
Your samples can stay unfrozen in fridge for up to 48 hrs. Otherwise freeze
1. Label 13 x 100 mm borosilicate glass vials w/code number & place in the
manifold rack (white plastic that fits into the glass manifold). Label the tubes
with white labeling tape and pencil and make sure that the labels are taped
(with Scotch tape) to the vial.
2. Only pour as much chemical as you will need so if you have 16 spe then you
just need 4ml x 16 water for example.
Activating the Column:
3.
4.
5.
6.
7.
8.
Put one LICHROLUT C18 column onto each ‘nib’ on the lid of the manifold
(again there is a maximum of 12). Open all of the stopcocks.
Pipette 2ml of 100% methanol (MeOH) into the top of each LICHROLUT C18
column - the tip of the pipette MUST NOT TOUCH THE WHITE SOLID IN THE
COLUMN. Some of methanol will sink into the column.
Make sure the valve used to regulate the vacuum on the manifold (a white,
moveable cylinder located near the gauge) is turned in the ‘vacuum’ position
(hole not open). Turn on the vacuum pump and draw the MeOH thru each
column and into the waste container. When all of the MeOH has passed
through the column, turn the vacuum off and ‘bleed the vacuum’ on the
manifold. To bleed the vacuum, turn the white, moveable cylinder to the
‘bleed’ position – line up open circle on the cylinder with open circle
underneath.
Pipette another 2ml of MeOH into the top of each column, turn the pump on
and draw the MeOH through the column. Turn the vacuum off and bleed the
vacuum. Leave a little fluid right above the white column
With pump off, pipette 2 ml distilled water into the top of each column, then
turn the pump on and draw the H2O through column. Turn the vacuum off and
bleed vacuum. Leave a little fluid right above white column
With pump off, pipette another 2 ml distilled water into top of each column,
then turn the pump on and draw the H2O through the column. Leave a little
fluid right above the white column. Turn the pump off. Turn the vacuum off
and bleed the vacuum.
Running the Samples:
9.
10.
Place the parafilm wrapped end of the CLEAN tygon tubing snuggly into the
opening of each column and the ‘bare’ end into the tube containing the water
sample. BE SURE TO MATCH THE WATER SAMPLE NUMBER TO THE CODE
NUMBER ON THE GLASS TUBE and C18 cartridge.
Turn on the pump and carefully monitor that all of the water from each sample
tubes goes through the column.
Gabor Hormone
11.
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a.
Make sure all water is pulled off of the column. When the samples are
completely done, turn the vacuum off and bleed the vacuum.
b.
Remove the tygon tubing from the LICHROLUT C18 columns and set
aside for cleaning.
c.
Take a 1-1.5 ml sample of waste water from either the waste container
(12-port manifold). This sample should be put in a labeled1.5 ml
eppendorf tube for analysis (to test for hormone leakage during
extraction). WE don’t do this but maybe we should
d.
Remove the waste container and discard the waste (12-port manifold)
and disconnect the tubing from the Erlenmeyer collection flask and
discard the waste (24-port manifold). Dry out the waste container
before putting in manifold rack.
12. If you are not ready to deal with SPE you can cover them and store them in -20
until ready to use. Defrost them before continuing.
13.
Now take the manifold rack with your labeled glass tubes and put it into the
manifold (the notch must go by the pressure gauge). Replace the manifold lid
making sure that the tip extenders from each column go into each of the
appropriate glass tubes.
14.
Pipette 2ml of MeOH into the top of each column. Turn on the vacuum pump
and draw the MeOH thru each column and into the glass tubes. Turn off the
vacuum and bleed the vacuum. Do this slowly to get most of the hormones. Be
sure that you just turn on the vacuum enough to start dripping but very slow
dripping so the methanol has time to bind to the hormones and drag them into
the test tube. If you run it through fast you might not catch all of the hormones.
15.
Pipette another 2ml of MeOH into the top of each column, turn the pump on
and draw the MeOH through the column. Do this slowly to get most of the
hormones . Turn off the vacuum and bleed the vacuum.
16.
When all of the MeOH has been collected in the glass vial, run the pump for 1
min extra to dry out the C18 tubes. Then turn off the pump, bleed the manifold
chamber, remove the lid and cap off (or parafilm) each vial.
17.
Store all unused SPE in storage bin with drying capsules/bags
18. Clean up room and rinse out all tygon tubing with water so nothing yuck is left
* If processing samples immediately, proceed to “Dehydrating the Samples” section
* If processing samples at a later date, store the capped vials with MeOH at 4C or 20C if it will be a while.
Nitrogen-drying of samples
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place samples in water bath @ 37ºC and use an evaporating manifold
connected to a nitrogen tank to push a gentle stream (10psi) of nitrogen over
samples until dry (1hr ish). All of this is in Horne lab. If water bath is not there
you need to borrow it from Horne
Be sure there is at least an inch of DI water in the water bath
Be sure the valve leading to the two ports is open to either both ports or just
one if you are not running a full plate of samples
Check every 15 min to remove dried samples. If not resuspending then put
dried sample in -20C…then proceed to resuspension step.
Clean the nipples in between samples with ETOH.
Gabor Hormone
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Note and Mark where we left off the Nitrogen. Let me know to order more if
we running out. It comes in on Thursdays so we need to order by Wed.
Resuspension of Steroids:
1. Make EIA buffer or check that it is not more than one month old (carefully squirt
water into bottle to clean it out fully when making buffer)
2. When you’re finished dehydrating (either put in -20 until ready or), immediately
resuspend the steroids in 5% EtOH, 95% EIA Buffer – the amount depends
on your sample. Say, you were resuspending the steroid pellet in 1000μl of
total volume. Add 50μl of 100% EtOH, vortex for a minute, then add 950μl of
the appropriate buffer and vortex. We usually use 17.5 ul EtOH and 332.5 ul
EIA buffer for mollies but at 1:2. We use 250 ul (we need to make it more
dilute) for toads, 400ul for salamander. Gambusia we use 350ul total too but at
1:! For free or conjugated we should use less total buffer as those numbers
will be lower. Maybe 1:1 for sailfin mollies.
3. Shake samples at 1500rpm for 1hr and put in fridge for MAX 1-2d before EIA.
If more than that, store at -20ºC then defrost completely.
4. Get your Excel plate file ready and determine dilutions. Print out plate on a
landscape and use to fill the plate. Be sure to pick the correct hormone from
top left box. Control should be first and last sample
5. Check the booklet for plate process so you know when to start
a. Kt = shake for 18 h at 4C
i. Dilute Pl and Pm 1:8-15
b. Cortisol = 16-18 hr at 4C no shaking according to book
i. Dilution for pl control = 1:20
ii. Dilution for most pl samples = 1:14-16
iii. Dilution about 1:8 for Gambusia
c. Corticosteroid = 2 hrs shake room temp
i. Dilution for frog Ao pool 1:10, Ao samples 1:14
ii. Dilute E. nana 1:1
iii. Dilute E. tonkawae 1:1
d. T = 2 hrs room
i. Dilute Pl and Pm 1:10
e. E = 1 hr room temp
i. Dilute Pl and Pm 1:4
6. Shake 4c samples in yellow foam with test tubes with black lids in four outer
corner at 1500 RPM for 1 hr right before running plate
7. If you need to dilute samples then label microcentrifuge tubes and put in
appropriate amount of EIA for dilution until the shaken samples are ready to
be added. Use C1V1=C2V2. Ex – you want 130 ul (you need this much to safely
get enough for duplicate run) at 1:10 and you started with 1:2 = 0.1 x 130 ul =
0.5 x V. V = 26 ul of sample plus 104 EIA buffer = 130 ul total volume
a. Shake whole microcentrifuge tube holder on vortexer
8. Get your control pool sample ready – what will you use for each plate that you
have plenty of?
9. Make standards – save the Bulk standard for future standards if this is first time for
5 kit box. Label and put in 4c
Gabor Hormone
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10. Make Tracer and Anti serum – clean out container carefully but do not shake –
just flip upside down and right side up. If this is 5 plate kit then put it into a
clean larger flat bottom glass vial and save on fridge door. Use 5.5 ul in white
tray of each, save rest
a. If it is 1 plate kit make it in the actual vial..
b. This is the most expensive part of the kit after the plate. They are
viable for only 4 weeks.
11. Fill plate carefully. Use same tip for standards. Use same method of equilibriation
of the tip for each well. Use multichannel pipette and one clean white tray for
tracer and other for antiserum except in first column. Clean all white trays
each time with Etoh.
12. Cover plate with all but edge of sticker and leave portion of white over the
letters. Save sticky cover after plate runs.
13. Use orbital shaker on #3 for appropriate amount of time for all but cortisol. Use
Mclean walk in cold room on 3rd floor for 4C.
14. Put all your samples away in -20C unless reusing in less than 48 hr.
15. Make Wash buffer if needed (lasts 4 weeks). Be sure you have fresh ultrapure
water to make this or it does not work well. Polysorbate 20 is viscous use 1000
pipette and suck up VERY slowly. If you fill squirt bottles cover end with
parafilm between uses. Note if big one or small one. Make sure there is
plenty of water left in room for others
16. Make Ellman in tinfoil bottle (note if 100dtn or 250 dtn to determine water amnt)
17. Dump plate liquid in sink
18. Use squirt bottle to rinse plate 5x (squirt into each well and do not mix liquid in
wells on first column). Dump and shake off each time
19. Dry plate on paper towel by taping it while it is upside down to get all liquid out
20. Use our Dark Grey Multichannel 200ul pipette to put Ellman from Ellman white
tray into all wells quickly.
21. Orbital shake at room temp on #3 for appropriate time
22. Us id access for Dr. Aron lab and turn on computer and Plate reader. Bring USB
reader
23. Start Gen 5 -> open experiment-> GaborLab (412 wave)
24. Start Excel
25. Make sure it will read – hit top 2nd to last button to read (<=)
26. Hit box with X to save file to Excel
27. Open Excel Plate file and fill with Excel sheet data
28. Bring plate and orbital shaker and put on same table as plate reader
29. Check that r2 as close to 1. If plate is not reading stnd 8 & maybe 7 may be
bigger Bo. So you need to mask this part to have a 6 or 7 standard line.
30. You can rerunplate if Bo is really high or plate is not running right
31. Save all stuff onto your own USB
32. Be sure to turn off the microplate reader and clean up
33. Go back to genetics lab and throw out standards (not the bulk) and clean up
34. Clean up Nice lab. Do not leave standards in fridge and put all back where they
came from. Put all our stuff into the box. Put EtoH on shelf with other
chemicals.