OXONO.N BioAnalysis MANUAL FOR NITROSO-THIOL KIT

Vial 1- Greiss A + cleavage reagent

Vial 2- Griess A

Vial 3- Griess activator

Vial 4- Griess B

Vial 5- Nitroso-glutathione standard

Vial 5a- for diluting nitroso-glutathione standard

1 96 well plate

Kit Manual

If any item is missing, please call us and ask for the Kit Support Team 510-215-7077 ext. 34.

Important Storage requirements:

Due to a recent innovation, we can guarantee the performance of our kit for up to one month even when kept at room temperature. When stored at 4^oC, the kit can be stored for up three months. Whatever temperature is chosen, we do recommend, for optimum performance, that the nitroso-glutathione standard (Vial 5) is kept at -20^oC in the dark.

Additional items required:

A plate reader with a 540nm filter

An adjustable pipettor

A repeating pipettor (suggested)

High purity water- ideally MQ+ (R Waters Inc) or HPLC grade.

General tips for optimal performance:

The lower the quality of water used, the higher the background will be. Tap water, for example, contains nearly 1mM nitrate. Dispose of pipette tip wastewater into a beaker: expelling waste into a sink can lead to splashback which would result in significant contamination.

Sample number:

This kit is for 96 analyses. This includes standards which must be run at the same time and in the same plate as unknowns. It is a matter of personal choice as to how many standards need to be run, however, we recommend a minimum of eight, run in duplicate. Thus, approximately

70-80 unknowns can be analyzed with one kit. Again, it is a matter of personal choice whether the unknowns are run in duplicate: if so then 35-40 unknowns can be analyzed with one kit.

Legal Strictures:

The kit is for research use only. It is not designed for clinical diagnostic purposes. Please take all appropriate care when opening the vials. We strongly recommend the use of eye protection and gloves.

Background:

The assay of nitrosothiols (RSNOs) appears to provide a very useful index of nitric oxide

synthase (NOS) activity. The concentration of RSNOs has been shown to increase with localized induction of NOS. Further, they have been shown to fall rapidly upon administration of NOS inhibitors.

Our kit provides a complete solution for the assay of RSNOs. It is very flexible, allowing the researcher (by use of simple ultra filters), to assay low and/or high molecular weight RSNOs. The assay is divided into essentially two steps; in the first the sample is prepared according to which subgroup of RSNO is desired. The S-N bond is then cleaved, resulting in the formation of an equivalent of nitrite which is assayed using the Griess reaction. The intensity of the color formed is linearly related to the concentration of nitrite and hence to the original RSNO concentration.

Sensitivity:

Sensitivity is determined by two factors: the fluid being analyzed and the detection method used. Plasma, for example, contains many substances which can interfere with the Griess Reaction. Consequently, only 40m l of plasma may be analyzed in a well; sensitivity is therefore reduced by approximately 60%.

For all other fluids: using a plate reader, sensitivity is approximately 500nM. However, if a microcuvette is used in a spectrophotometer, then up to a five-fold increase in sensitivity can be obtained.

Tips for Optimal Performance:

The most significant factors affecting the performance of this Kit are pipetting accuracy and background interference. We recommend the use of a repeating pipettor since this increases the precision of the delivery into the wells. Secondly, nitrate and nitrite are everywhere present; beakers, pipette tips, water, hands etc. Washing all plastic and glassware with high purity water will significantly improve the results. In cell and tissue culture work, a very significant contaminant is the nitrite present in culture media. This results in a typical background of 2-3m M nitrite. OXONON has pioneered the production of reduced-nitrite-containing media. Our media contain a background of 400-600nM nitrite-- a significant advance when one is trying to measure a typical 200nM increase in NO/nitrite, after cell stimulation.
 

Overview:

The chemistry of the Kit is based on two classical chemical reactions: Griess and Saville. In essence, a cleavage reaction breaks the S-N bond of nitroso-thiols. This results in the release of an equivalent of NO which in turn gives an equivalent of nitrite. The nitrite is detected colorimetrically using the Griess reaction. It is a very precise and accurate method for measuring RSNO concentrations, however a potential problem is the presence of nitrite in the samples. For this reason, we include a second Griess A vial (Vial 2) which does not contain the cleavage reagent. and therefore only detects free nitrite. One may simply subtract Vial 2 values from Vial 1 values to ascertain the contribution from RSNO. This is only necessary in situations where nitrite concentrations are a significant percentage of RSNO concentrations.

A typical analysis requires a group of wells from the 96 well plate to be used for a standard curve. In this Kit we supply a known standard of nitroso-glutathione which may be added or spiked into replicate samples, or water if no spare sample is available. This will allow for the generation of a standard curve to determine the unknowns. We highly recommend that you run the standard curve in the bio-fluid you are analyzing. However, if this is not practical, you may dilute the GSNO into water as shown below. So, the first step is to set up the standard curve as follows:

How To Set Up The Nitroso-glutathione (GSNO) Standard curve.

Add 1000m l of ultra pure water to Vial 5 and mix well; take 10m l of this solution into 1.49 ml of water in the clean unlabelled vial and mix well. Label this vial as Vial 5a - this is your Master solution. Keep this solution at 4° C and wrapped in aluminum foil. Use ideally within 1-2 hours as it slowly decomposes when dissolved in water. Include at least seven standards from the following table in your sample set. Also, add 200m l of water to two wells for use as blanks for the plate reader.

Generate a GSNO standard curve: Pipette out from Vial 5a as follows into wells designated as standard wells; then add ultra pure water as indicated in the second column:

m l Vial 5a m l bio-fluid/water Final concentration GSNO m M

0 100 0

2.5 97.5 0.25

5.0 95 0.5

10 90 1.0

20 80 2.0

30 70 3.0

40 60 4.0

60 40 6.0

100 0 10.0

Samples: For total (i.e. high and low Mwt) RSNO, simply add 100m l of sample into wells designated for unknowns. Then immediately follow the Step 2 process outlined on the next page.

For high or low RSNOs, simply ultrafilter the sample using an ultra free filter (e.g. Millipore). Then, either transfer 100 m l of the filtrate to a well for low Mwt RSNOs or add water to the retentate so as to bring it back to the volume applied to the filter, and use 100m l of that for high Mwt RSNOs. Once prepared follow Step 2 on next page.

Once the plate is set up, proceed to Step 2 as quickly as possible.

NOTE: For plasma analysis, a slight modification is required. Firstly only 40 m l may be analyzed--dilute to 100m l with ultra pure water in the well. Further, older or multiply frozen plasma tends to give rise to colloidal samples during the analysis which artificially increases absorbency reading. To reduce this effect, spin the samples at 5000g for 10 minutes prior to analysis. Better, use an HPLC type 0.22m m filter to remove particulate matter. This effect is rarely seen in fresh plasma.

Prepare Griess Reagents:

Add 0.4ml from Vial 3 to Vial 1, and 0.4ml from Vial 3 to Vial 2. Gently mix well and with care! Store on ice.

Add Color Generating Reagents (Griess)

Add 50m l from Vial 1 to each well, followed immediately by 50m l from Vial 4 to each well. A purple color should be visible. Wait 10 minutes, zero the plate reader against the water-only blanks and then read the plate at 540nm to obtain the absorbency values for each well.

Calculate the results

Plot the absorbency of the known standards against concentration; this will be a straight line. The concentration of the unknowns can then be read off the standard curve.

Control Reagent (Vial 2):

Since high background levels of nitrite in the samples will give rise to color formation in addition to those of nitrosothiol concentration, we supply Vial 2 which is Griess A but without the cleavage reagent. Should you wish to check that the reading you are getting is truly derived from a nitrosothiol, simply re-analyze the sample in duplicate. To one add 50 m l of Vial 1 as before, and to the other instead add 50 m l of Vial 2. The difference between the first and second sample will indicate the amount of signal deriving exclusively from RSNO. If the changes in RSNO are expected to be small relative to nitrite concentrations, you may wish to analyze all your samples in this way.

Potential problems:

If you experience any difficulty please try the following remedies. If, however, you are still having problems, please call our Kit Team at 1-510-215-7077. (9.00 am to 11.30 am PST).

1. Poor Precision:

It is undoubtedly due to poor pipetting technique or particulates/bubbles in the wells. Try using a repeating pipette for optimal accuracy. In our studies, the Kit assay has a very low cov (<5% in the mid-range of the standard curve). Also, try coating each tip with the reagent or sample to be pipetted- i.e. take up the required volume and expel back into the vial, repeat and then dispense the required volume into the well. This really helps precision.

2. No color!

Have you added the color reagents? (Vials 1 & 4). If any of the reagents were omitted, you unfortunately will have to repeat the whole analysis.

3. High Background?

Check a few of your samples with Vial 2 as detailed above-- you may have high concentrations of nitrite in your samples.

Feedback:

Our intention is for you to have great results with this Kit, so please write or call if you should experience any difficulties. We have many years of experience in measuring nitric oxide and invite you to take advantage of this expertise to ensure optimal results for your study.

Contact our Kit Team on: 1-510-215-7077 or email us: OXONONUS@aol.com

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