sulfate testing hacks

Biosystem’s Sulfate test is meant for 96 well plates… This link says that it is good for plasma and urine. It is a wonderful 96 well assay that requires a plate reader. This is the way to go if one wants to run several dozen tests at a time. It requires the allotment of wells in the 96 well plates for know standards so that one can create a linear calibration curve. This can be a pain and a time drain. The good part of this test is that comes with trichloroacetic acid TCA with it that allows the user to precipitate proteins out of the biological fluids before adding the barium… that forms a precipitate with the sulfate. Overall, this seems to be a very nice test for everyone but those just analyzing a few samples at a time.

There is the protocol for BioAssay sulfate…

KIT CONTENTS (200 tests in 96-well plates) Reagent A: 25 mL Reagent B: 2.4 g Powder
TCA Reagent: 25 mL Sulfate Standard: 1 mL 60 mM

Urine samples should be diluted 10-fold in deionized water prior to assay.
Fresh serum or plasma (non-hemolyzed) samples can be either assayed immediately, or frozen for future tests. Samples should be deproteinated as follows: mix 200 μL sample and 100 μL TCA Reagent in a 1.5-mL Eppendorf tube. Spin down protein precipitates 5 min at 14,000 rpm on a table centrifuge. Transfer 200 μL supernatant for assay.

“Prepare enough Working Reagent for all samples and standards (100 μL WR per
assay well) by mixing 95 mg Reagent B per mL Reagent A, i.e. 10 assay wells, mix 95 mg Reagent B with 1 mL Reagent A. Vortex for at least 1 min to ensure complete dissolution of the powder and incubate the reconstituted Working Reagent for 10 min before use.
Use a multi-channel pipettor, add 100 μL Working Reagent to each assay well. Tap plate to mix well. If TCA precipitation was required, mixing the samples and standards with the WR can be improved by pipetting up and down once. Incubate 5 min at room temperature and read optical density at 540- 610nm (600nm).”

Here we are left to assume that the powder is some sort of barium salt. The solution might contain some sort of buffer. Probably the important part is the mixing if TCA was used.

A quick paragraph on TCA.

TCA is widely used to precipitate proteins from solutions. According to Wikipedia and what is sold on Ebay, it is also used for protein precipitation and removal of warts and related skin lesion. In my lab days we started off with a saturated solution, that may have been 80% as sold on Ebay:

In comes the eXact meter hat has software already in it calculate concentration based on prior experience on how these tests perform. This can save the lab novice tons of time and frustration.

How to precipitate proteins with ~ 80% TCA

This protocol comes from the proteomics lab at KU Medical center.

Reagents

Preparation of 100% TCA: (don’t try to weigh out TCA; it’s too hygroscopic)

• Obtain a fresh bottle of crystalline TCA. oEbay does not seem to sell such things. Such powder might be sold in specialized cosmetaology stores that cater the the beauty industry. The powder will save a lot of money.
• Read the weight in the container from the label.
• Add distilled water to give a 100 g / 100 ml solution at final volume.

Procedure:

1. Add 100% (w/v) TCA (trichloroacetic acid, ( see preparation method above) to the sample to bring the TCA concentration to 20%. In other words, one part 80% TCA to three parts urine, plasma, or other protein containing solutions.
2. Incubate on ice for at least 1 hr. Diluted samples may be left overnight.
3. Spin at maximum speed in a microcentrifuge for 10 min. In this case, a clinical centrifuge.
4. Wash the pellet 3X with a solution of ice cold 0.01 M HCl / 90% acetone. Allow the pellet to air dry. Skip this step. The interest is in the supernatant, or top layer.
   The pellet can then be resuspended directly in 100 mM Tris, pH 8.5, 8 M urea or 6 M Guanidine- HCl in 25 mM ammonium bicarbonate buffer for enzymatic digestion for mass spectrometry There might be some interest in keeping the pellet as it contains proteins that might have some research interest.

The eXact Meter

The eXact meter works with a smart phone. FiltersFast.com sells test strips for the photometer. Customer service was called. This instrument works with some other organic acids. It might help to have some dilute sodium hydroxide, NaOH, Draino, on had to adjust the pH.

Recall that the 96 well plate BioAssay dit callys for pipetting up and down if TCA was used. It may be a good idea to push the strip back and forth a few times. It might also help to rinse out the cell after use.

Purists will turn up their noses at not having a calibration curve for each and every measurement. Sure, something in the sample could interfere with the turbidity of the barium sulfate precipitation that the instrument is measuring at the assigned wavelength. There are a number of cheap forms of sulfate, like calcium sulfate (Gypsum) that can be obtained from Garden stores. Ebay sells potassium sulfate that is reasonably priced.

The potassium sulfate can be used to make stock solutions to test the hypothesis hat junk solutions other than water are interfering with the test. A mock TCA precipitation can be performed on such a solution. Does the eXact meter give the same result? If not, by how much is it off?