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.
Clean nibs with ethanol. 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.
4.
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.
5.
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.
6.
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
7.
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
8.
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 water Samples:
9.
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.
10.
Turn on the pump and carefully monitor that all of the water from each sample tubes goes
through the column.
11.
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
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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
 place samples (66?) in white test tube rack (note x where it doesn’t work) 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
 Clean the nipples in between samples with ETOH.
 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
 First open the main valve on the tank, then the side valve on the regulator just enough to
hear air come through. Then use the lower valve on the regulator to regulator the low
flow that has been turned on from the side valve. When done close all valves in the same
order
 Check every 15 min to remove dried samples. Push down manifold and turn nitrogen up
after first 15 min but not thereafter
 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.
 If not resuspending then put dried sample in -20C…then proceed to resuspension step.
Ryan Earley says to resuspend the same day
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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 but better to not),
immediately resuspend the steroids in 5% EtOH, 95% EIA Buffer – the amount
depends on your sample. We usually use 17.5 ul 95% EtOH and vortex for 1 min on
multitube vortexer then add 332.5 ul EIA buffer for mollies but at 1:2 . We use 250 ul
(we need to make it more dilute) for toads, 260 ul for salamander. Gambusia we use
350ul total too but at 1:1 For WATER sample use either 260 ul or 130 ul if you think it
needs to be very concentrated and get two samples. For free or conjugated we should use
less total buffer as those numbers will be lower.
3. Shake samples at 1500rpm for 20-30min and put in fridge a day before plating. If more
than that, store at -20ºC then defrost completely.
3. Get your Excel plate file ready and determine dilutions. Always download fresh plate file
to prevent cut and paste error from Cayman. Use double not triple
https://www.caymanchem.com/app/template/analysis%2CEIA.vm
4. 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 or both last samples
5. Check the booklet for plate process so you know when to start
a. Kt = shake for 18 h at 4C
i. Dilute Pl female 1:6 pl Male: 1:16-20
ii. Dilute Pm 1:4 females and 1:6-8 males
b. Cortisol = 16-18 hr at 4C no shaking according to book
i. Dilution for pl control = 1:20
ii. Dilution for most pl juvenile = 1:14-16 , pl adult ~1:75
iii. Dilution about 1:8 for Gambusia
c. Corticosteroid = 2 hrs shake room temp
i. Dilution for frog Ao n pool 1:10, Ao samples 1:14
ii. Dilute E. nana 1:1
iii. Dilute E. tonkawae 1:1
iv. WATER sample: 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. Day of shake samples for 2 hrs
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. Make sure samples from freezer are placed on ice – maybe even ones from fridge to maintain
temp since the room is warm
9. Get your control pool sample ready – what will you use for each plate that you have plenty
of? Take out small microcentrifuge vials of this instead of your main pool that will be in
a testtube so that you don’t get freeze thaw issues. Always keep it on ice
10. Make standards –
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11. 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 5.4 ml 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.
12. Make sure to properly mix EIA buffer before you put it on the plate. Do not let other stuff get
into it so cover it
13. The Bo wells in the first row are your most important points for the plate. Make sure you
shake EIA and equilibrate pipet before each well to make sure all three are as close to
each other as possible.
14. Use same tip for standards. Use same method of equilibriation of the tip for each well.
15. Vortex each sample for 10 sec before putting on plate including pool sample. Do not vortex
for hour day of anymore
16. 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.
17. Cover plate with all but edge of sticker and leave portion of white over the letters. Save
sticky cover after plate runs.
18. 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.
19. Put all your samples away in -20C unless reusing in less than 48 hr.
20. 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. Only make
2 liters at a time (check size of bottle to make sure what quantities are needed)
21. Make Ellman in tinfoil bottle (note if 100dtn or 250 dtn to determine water amnt)
22. Dump plate liquid in sink
23. 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
24. Dry plate on paper towel by taping it while it is upside down to get all liquid out
25. Use our Dark Grey Multichannel 200ul pipette to put Ellman from white tray into all wells
quickly.
26. Orbital shake according to book directions on #3 for 60 min
For plasma samples
1. Blood maintained on ice until centrifugation at 17 x10 rpm for 5 min or 3000 g for 10
min to separate blood from plasma.
2. Dilute measured amount of plasma in 4 ml distilled water followed by the solid phase
extraction procedure using Sep-Pak® Plus C18 (methanol (total) or ethyl acetate (free)
and methanol)
3. Extracted plasma samples re-suspended in ? μl x original plasma volume or somewhere
there in.
4. Final concentration is plate pg/ml / dilution. Ex. 360 pg/ ml / (10ul plasma/400 ul EIA) =
1440pg/ml. or 360pg/ml X 40x dilution = 1440pg/ml
Using New BioTek ELx800 Plate Reader
***Turn Plate Reader off and keep covered when not in use***
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Creating New Experiment:
1. Turn Plate Reader on and wait until screen flashes "Ready".
2. If plate reader screen does not say "Ready", press the Main Menu button on keypad and
that should get the plate reader ready.
3. Open program "Gen5 2.05" on desktop computer
4. When program opens to default window (Task Manager), click "Experiments" on sidebar
menu.
5. Then click "New..." option
6. In left sidebar menu, double click "Procedure" under Protocol
7. In pop-up window titled Procedure, click "Read" under Actions in left sidebar menu
8. Change Wavelengths option to 405 in dropdown menu. Click "OK". Then click "OK"
again.
9. Double click on "Plate 1" in sidebar menu, then click giant icon of a "green play button"
on top menu to read plate.
10. Place plate with well A1 in top left-hand corner (when facing plate reader). Click "OK"
when ready to read.
11. To export into excel, click button that has excel symbol on it.
12. If you need to read another plate, click on "green play button" and it should create a new
plate. OR, you can re-read your Plate 1.
13. To delete and re-read your Plate 1, click on "Plate" located at top menu. Then select first
option "Delete data of Plate 1 and re-read".
Adding to your Existing Experiment:
14. Turn Plate Reader on and wait until screen flashes "Ready".
15. Open program "Gen5 2.05" on desktop computer
16. When program opens to default window (Task Manager), click "Experiments" on sidebar
menu.
17. Then click "Open..." option and double click whichever experiment you want to open.
18. Follow above instructions to create a new plate or re-read any old plate.
19. Hit box with X to save file to Excel
20. Open Excel Plate file and fill with Excel sheet data
21. Reading plate - In the Subtracted section:
Look at Bo and see that the three numbers are similar and then look at average max binding Bo
and make sure standards are below that. For the TA look that number is higher than Bo values. If
one of two standards is off you either drop the standard or put same number twice for better
value.
22. At bottom left of page look at range of 20-80% to make sure our diluted values are in that
range as those are best reads.
23. Pick lowest variance. 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. Or Cut
and paste value from sample that worked best.
24. You can rerunplate if Bo is really high or plate is not running right
25. Save all stuff onto your own USB or load it onto DROPBOX now that we have internet
26. Be sure to turn off the microplate reader and clean up
27. Clean up lab. Do not leave standards in fridge and put all back where they came from.