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Product Details
| Cat # +Size | K555-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Absorbance (560 nm) |
| Species Reactivity | Mammalian |
| Applications | The assay is useful over the range of 0.1-2 μg. |
| Features & Benefits | • Simple procedure; takes less than ~100 minutes • Convenient and easy to use |
| Kit Components | • Oxidation Buffer • Chloramine T Concentrate • Perchloric acid/Isopropanol Solution • DMAB Concentrate (in DMSO) • Hydroxyproline Standard (1 mg/ml) |
| Storage Conditions | 4°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Hydroxyproline (4-hydroxyproline) is a common nonproteinogenic amino acid. It is found only in collagen and elastin in mammals but exists in a number of other proteins in plants. Hydroxyproline is formed only as a post-translational modification in the peptide chain and proline hydroxylase does not hydroxylate free proline. Hydroxyproline in tissue hydrolysates is a direct measure of the amount of collagen or gelatin present. A variety of disease states are believed to affect collagen turnover and can cause elevated serum or urine hydroxyproline. Such conditions range from neoplastic, inflammatory, renal or bone disease to endocrine and autoimmune disorders. BioVision’s Hydroxyproline Assay Kit is designed to measure hydroxyproline in tissue or protein/peptide hydrolysates. It can be used to measure hydroxyproline from other biological samples such as serum or urine if they have undergone a prior purification process. It is an easy, convenient method which results in a chromogen with an absorbance maximum at 560 nm. The assay is useful over the range of 0.1-2 μg.
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In the protocol for serum or urine, after hydrolyzing the samples, you need to add activated charcoal. This step is absent with the tissue samples. Is this a mistake?
Charcoal treatment is to clarify the samples, which is not needed for tissue samples.
After incubation with DMAB for 90 min at 60 °C, how soon should I read the plate? Can I wait for a few hours before reading the plate?
After incubation with DMAB for 90 min at 60°C, the plate should be read immediately when it is still warm. This is because DMAB has the tendency to precipitate and settle at the bottom of the plate if waited longer.
What vials would you recommend for the hydrolysis step?
You can Polypropylene vials (Fisher #03-313-82B) and with screw caps without O-ring (Fisher # 03-390-55)
What kind of purification is needed for Urine sample?
To 100 ul of serum/urine, add 100 ul of conc. HCl in a pressure-tight, Teflon capped vial and hydrolyze at 120C for 3 hrs. Add 5 mg of activated charcoal Norit or activated charcoal Darco (Sigma Cat# 97876 or 242276) & mix. Centrifuge at 12000g for 2 min. Transfer 10 ul of supernatant to 96 well plate & evaporate to dryness under vacuum. Proceed ahead with regular protocol starting from step 3.
Should the Hydroxyproline Standard also go through the drying process as hydrolzed samples? If it is possible, could you tell me the reasons why the hydrolyzing process is required?
The samples are being digested/hydrolyzed with acid to calculate the hydroxyproline from the collagen. The standard IS HYDROXYPROLINE so it does not need the HCL/cooking treatment. In addition, the standard is NOT vacuum dried because it does not contain HCl-the samples are dried to remove the HCl before proceeding with the other reagents.
In the manual it says to dry the plate under vacuum. Since I don't have the equipment readily available for this, I was wondering if the plate could be dried in an oven at, for example, 37 degrees Celcius or just to air at room temperature
You can dry the plate in an oven at 60C make sure to get rid of the HCl. Air drying will take a very long time and is not recommended.
Is it possible to keep the hydrolyzed lysate or the dried 96-well plate for continuation at a later time? If possible, what are the storage conditions?
Yes, you can store either / both the hydrolyzed sups and the dried plate at -80 degrees C for later use. Please aliquot the lysate and seal the plate tight before storing.
Is it is acceptable to reduce the reaction time in sample preparation part, 120.C, 3 hours? If yes, how long should be done at least?
The hydrolysis will not be completed efficiently if done for lesser than 3 hrs. If your concern for the time is that you would not be able to finish the whole expt the same day, you can always hydrolyze O/N as well and continue the next day, or hydrolyze for 3 hrs and freeze the hydrolyzed sup at -80C in aliquots for use the next day.
My cells gave a burnt appearence. (this step, To a 100 ul sample of homogenate, add 100 µl concentrated HCl (~12N, not provided) in a pressure-tight, teflon capped vial and hydrolyze at 120C for 3 hours.) Is it normal?
The charred or burnt appearance is not a problem. It is expected that when you subject cell/tissues to that high a temperature for 3 hrs they give the burnt appearance. Before you proceed to the next step, let this black substance settle down as a debri (give a gentle spin to the tubes if required). Then take only the clear supernatant for the next step of evaporation. I suspect that some of the charred tissue is going into your analysis wells in the duplicates and hence you are getting variable results. If you take just the clear sup, you should be able to circumvent this problem.
Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kit's citations to see what kind of samples have been used with this kit other than mammalian samples.
Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Can we use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact yopur regional BioVision distributor.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20C. Please refer to the datasheet for storage information and shelf life of each of the components.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
How do I normalize my samples against protein concentration?
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, I would strongly recommend you to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.
How is K555 different from K226
K555 follows acid hydrolysis protocol where as K226 follows alkaline hydrolysis and it is percholate free. They differ in the method used to hydrolyze the tissue. Alkaline hydrolysis is less time consuming than acid hydrolysis.
How can I make sure that sample hydrolysis is going well? How will the samples look at the end of 3 hour incubation?
At the end of 3 hours of hydrolysis, which is pretty sufficient for most samples, samples should be less viscous and more liquid like and can be easily pipetted unlike a thick homogenate. You may also see dark particulate matter (they are the lipids) settling at the bottom.
Uri Simcha Soiberman, Small Molecule Modulation of the Integrated Stress Response Governs the Keratoconic Phenotype In Vitro. Invest Ophthalmol Vis Sci, Aug 2019; 31390655.
Giovanni Ligresti, CBX5/G9a/H3K9me-mediated gene repression is essential to fibroblast activation during lung fibrosis. JCI Insight., June 2019; 31095524.
Yo Kishimoto, Reversal of Vocal Fold Mucosal Fibrosis Using siRNA against the Collagen-Specific Chaperone Serpinh1. Mol Ther Nucleic Acids., April 2019; 31100613.
Xuelian Xiong, Mapping the molecular signatures of diet-induced NASH and its regulation by the hepatokine Tsukushi. Mol Metab, Feb 2019; 30595550.
Nguyen Thi Thanh Hai, Selective overexpression of cytoglobin in stellate cells attenuates thioacetamide-induced liver fibrosis in mice. Sci Rep, Dec 2018; 30552362.
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