General

Q: Do I have to order via distributors?

A: You can order directly from QuickZyme, either through the website, or by sending an e-mail to ‘order@quickzyme.com’. However, in some countries or for some customers it may be easier to order via a distributor. For that reason we have a world-wide network of distributors (see website).

Q: Can I pay with credit card?

A: Yes, you can either pay by electronic bank transfer using the IBAN and BIC/Swift codes on the invoice, or you can pay by credit card. In the latter case we will send you a payment request through PayPal with instructions.  For such PayPal payment you don’t need a PayPal account; just a credit card is sufficient.

Q: Do you have trial sizes of your kits?

A: It is our experience that trial sizes are always too small to perform a useful pilot experiment. Therefore we do not offer trial sizes but offer low prices for high quality products.

Q: What is the shelf life of a kit?

A: Most kits have been optimized such that they have a shelf life of at least one year when stored according instructions.

Q: Do collagen assays contain recombinant proteins?

A: No, the total and soluble collagen assays contain purified rat tail collagen as a standard. The hydroxyproline assay uses the iminoacid hydroxyproline as a standard.

Q: What is the matrix effect?

A: Matrix effect includes interference between your assay and components in your sample. This often results in erroneous results. The matrix effect can be attributed to e.g. phospholipids, carbohydrates and metabolites, but also pH, viscosity, salt concentration or direct interaction between your analyte of interest and other components in the sample. It occurs with all kinds of biochemical assays such as GC, HPLC, ELISA or fluorescent and/-or colorimetric biochemical assays. When performing an assay with a new sample type for the first time, it is important to check whether matrix effect is present and how to prevent it. How do you know whether there is matrix effect? One way to determine if there is interference between your assay and your sample is to spike a known amount of the standard (e.g. hydroxyproline)  into your sample and compare the spiked sample with the unspiked sample; if the signal in the spiked sample is lower than the calculated value (signal unspiked sample + signal spiked standard) a matrix effect may be apparent. Another way is test various dilution of your sample: if no expected reduction of signal is observed upon dilution there is a matrix effect.

Q: How can I prevent a matrix effect?

A: The most common approach for accounting for matrix effects is to build a dilution curve of your sample (or a spiked sample if the signal is low) and see at what dilution the signal reduction correlates with the dilution. At a dilution where a linear correlation is observed it is safe to do your analysis for all your samples of the same sample type. For each new type of sample a new dilution curve analysis has to be performed.

 Total collagen assay/Hydroxyproline assay

Q: What is the difference between total collagen assay and the hydroxyproline assay?

A: The Total Collagen assay and the Hydroxyproline assay are quite similar, based on the same chemistry. The Hydroxyproline assay is for customers who want to measure hydroxyproline or have their own hydrolysis protocol; therefore it does neither contain a protocol nor tubes for hydrolysis, and it provides a hydroxyproline standard instead of a collagen standard. In the Total Collagen assay a collagen standard and a protocol and tubes for hydrolysis are provided.

Q: What is the species specificity of the total collagen assay and what types of collagen are recognized?

A: The total collagen assay can be applied for samples from all mammalian species. The assay does also not discriminate between different collagen types; all types of collagen containing hydroxyproline are measured. The kit standard used is rat tail collagen, which has a hydroxyproline content of 13%. Therefore,  absolute levels for specific collagen types with aberrant hydroxyproline content, such as in non-mammalian species (e.g.fish), cannot be assessed absolutely. However, relative amounts (compare groups, look for induction etc.) can be determined.

Q: After hydrolysis black particles are observed. Do they influence the assay?

A: After hydrolysis small brownish or black particles may be present. These particles result from carbohydrate and fat present in the samples. The amount of particles is very much dependent on the tissue type used for hydrolysis. After centrifugation some particles will float in the liquid. Upon transferring the liquid to a clean tube or plate, try to avoid those particles as much as possible. They do not interfere with the chemistry of the assay, the particles will only interfere when they block the light path in the photometer (usually a very narrow beam in the center of the well).

Q: Is acid hydrolysis for 20 hrs at 95°C complete?

A: Yes,  acid hydrolysis for 20 hrs at 95°C results in complete hydrolysis. This temperature also makes usage of a heat block possible, ensuring a more constant temperature.

Q: Can I also use hydrolysis protocols which hydrolyze at 110-120°C?

A: Yes, you can also use standard hydrolysis protocols using a temperature of 110-120°C. However, in that case we recommend not to use the Sarstedt tubes provided in the Total Collagen Assay kit but use your own (glass) hydrolysis tubes. Our observation is that regularly ovens show a high variability in temperature during o/n incubation (ranging from 100-140°C). At high temperature the Sarstedt tubes provided in the Total Collagen assay may leak giving variation in the results.

Q: Can I use an oven for hydrolysis?

A:Yes, you can use an oven for o/n hydrolysis. However, as described above, our observation is that regularly ovens have a very inaccurate temperature control and may show large temperature variations. It is advised to check the temperature with an external thermometer. At high temperature (>110oC) the Sarstedt tubes provided in the Total Collagen assay may leak giving variation in the results.

Q: Can I use a heat block for the hydrolysis step?

A: Yes. Since the hydrolysis step can be performed at 95°C, heat blocks can be used and are even advised since they hardly show any temperature variation.

Q: Sometimes my vial with buffer A is solid. Is it still OK?

A: Buffer A is based on DMSO. Since the melting point of DMSO is just below room temperature, it can become solid at lower temperatures. Just melting in warm water before use is enough.

Q: What dilutions should I use for various types of tissue?

In general, when using new tissue type samples it is advised to first test which optimum dilution is needed. The dilution of tissue extracts needed for optimal analysis depends on the type of tissue, and the amount of collagen present. For some tissues (mouse tissues, fish) we have published an application note giving directions for collagen content and sample dilutions (Application note: How to measure collagen in mouse tissuesCollagen analysis in fish tissue).

Q: In what buffer should I perform hydrolysis and make dilutions?

A: Dependent on the type and amount of tissue, add 6M HCl to the tissue to obtain 50-300 mg tissue/ml. A minimum volume of 100 μl is advised for hydrolysis. After hydrolysis add 0.5 volume water to 1 volume hydrolysate (e.g. 200 µl hydrolysate + 100 µl water). The sample is now in 4M HCl. All further dilutions should be performed using 4M HCl. 35 μl of the diluted hydrolyzed sample is used for analysis in the assay.

Q: How to store hydrolysates (temperature/how long)?

A: Hydrolysates can be stored for at least two weeks at 4°C or at RT. It is important that the tubes are well closed to prevent evaporation

Q: Can I concentrate my samples if collagen levels are low?

A: Many tissue samples such as lung, skin and vessels have high collagen content and do not need concentration. Tissue samples with low collagen content, such as healthy liver, cannot just be concentrated due to matrix effect. Biological tissue show matrix effect, which consist of factors disturbing the assay. Therefore many biological samples need dilution to be analysed in a good way. This is also the case for conditioned culture media. For mouse tissues we have published an application note which gives indications for dilution factors for various tissues.

Q: What is the matrix effect?

A: Matrix effect includes interference between your assay and components in your sample. This often results in erroneous results. The matrix effect can be attributed to e.g. phospholipids, carbohydrates and metabolites, but also pH, viscosity, salt concentration or direct interaction between your analyte of interest and other components in the sample. It occurs with all kinds of biochemical assays such as GC, HPLC, ELISA or fluorescent and/-or colorimetric biochemical assays. When performing an assay with a new sample type for the first time, it is important to check whether matrix effect is present and how to prevent it. How do you know whether there is matrix effect? One way to determine if there is interference between your assay and your sample is to spike a known amount of the standard (e.g. hydroxyproline)  into your sample and compare the spiked sample with the unspiked sample; if the signal in the spiked sample is lower than the calculated value (signal unspiked sample + signal spiked standard) a matrix effect may be apparent. Another way is test various dilution of your sample: if no expected reduction of signal is observed upon dilution there is a matrix effect.

Q: How can I prevent a matrix effect?

A: The most common approach for accounting for matrix effects is to build a dilution curve of your sample (or a spiked sample if the signal is low) and see at what dilution the signal reduction correlates with the dilution. At a dilution where a linear correlation is observed it is safe to do your analysis for all your samples of the same sample type. For each new type of sample a new dilution curve analysis has to be performed.

Soluble collagen assay

Q: How specific is the soluble collagen assay?

A: The soluble collagen assay is based on binding of Sirius Red to collagen. Sirius Red can interact via its sulfonic groups with various collagen molecules, leading to aggregates which can be precipitated by centrifugation. Other proteins that interact with collagen as well as high concentrations albumin may co-precipitate. The QuickZyme soluble collagen assay has been developed such, that non-specific precipitation is minimized (up to 5% serum no cross-reactivity of albumin occurs).

Q: Can higher amounts of sample be used instead of the 140 µl mentioned in the protocol?

A: The assay is based on a 96-well plate format. That limits the sample volume to 140 µl. However, larger volumes can be used but in that case centrifugation has to be performed in Eppendorf tubes instead of on a plate. If you use higher samples volumes you have to keep the same dye solution volume/sample volume ratio (60 µl dye solution/140 µl sample).

Q: Can I store the kit at room temperature?

A: The QuickZyme soluble collagen assay kit should be stored at 4°C. The collagen standard (rat tail collagen) should never be exposed to higher temperatures since it starts to denature above 25-30°C into gelatin. Denatured collagen / gelatin is not recognized by the soluble collagen assay.

Q: Do I need additional materials (buffers, solutions) to perform the assay?

A: In general the QuickZyme assays contain all buffers and standards needed to perform the assay. For the QuickZyme soluble collagen assay only special equipment is needed.

Q: What special equipment do I need to perform the assay?

A: The assay needs standard equipment present at most biochemical labs: a microplate reader capable of measuring at 540 nm and a refrigerated centrifuge equipped with microplate swing out carrier.

MMP activity assays

Q: What is the species specificity of the MMP-9 activity assay?

A: QuickZyme has two MMP-9 activity assays available: The human and mouse MMP-9 activity assays. Both assays are specific for MMP-9. In both assays human, mouse and rat MMP-9 are recognized. The human MMP-9 activity assay contains a human MMP-9 standard and has been optimized for human MMP-9, and the mouse MMP-9 activity assay contains a mouse MMP-9 standard and has been optimized for mouse MMP-9.

Q: Do the activity assays cross-react with other MMPs?

A: The QuickZyme MMP activity assays are specific for the particular MMP the kit is developed for and do not cross-react with other MMPs or other proteases. Specificity is introduced at four levels: the substrate, the capture antibody, the activation conditions and the buffers.

Q: What type of samples can be used?

A: Both tissue extracts and all types of body fluids such as plasma, serum, saliva and urine can be used. Protein denaturing reagents (such as urea and GuHCl) and reagents disrupting protein-protein interactions (such as SDS which prevents binding of MMP to the antibody) should be avoided. Also high concentrations of EDTA interfere by  inhibiting MMP activity by chelation of the Zn2+ ion in the catalytic site of MMPs.

Q: I have samples containing high and low MMP-9 concentrations; can I use them on one plate?

A: Yes, one of the big advantages of the MMP activity assays is the broad dynamic range. The measurement is not based on an end-point read-out but  the same plate can be measured after different time intervals. After  1-2 hours high activities can be measured, while very low activities can be measured after 6-24 hrs incubation. Background activity ultimately limits the useful incubation time.

Q: What does it mean that one can analyze both active and total MMP activity?

A: MMPs in biological samples can be present in various forms: as proMMP, as active MMP and as MMP/inhibitor complex (e.g. with TIMP). In general most of the MMP is present in its pro-form, which in nature can become active upon activation by other proteases. Therefore it is interesting to be able to measure both the active and the pro-form. This is integrated in the MMP activity assay kits. The same sample can be put in two different wells of the plate. After capture and washing in one well substrate is added and the already active MMP will be measured. In the other well a pre-incubation with APMA is carried out, which is able to activate the proMMP in vitro. Following this pre-incubation, substrate is added, resulting in the measurement of both already active MMP as well as APMA-activated proMMP (= total MMP). Thus, using one assay a double amount of information can be extracted from one sample.

Q: Why do I need to add APMA to measure total MMP activity?

A: As described above, most of the MMP is present in its pro-form. In vitro, proMMP can be activated by APMA, a mercuric compound that interacts with the SH-group in the pro-domain of MMPs and such preventing activity (Sellers A et al., Biochem J. 1977, 163(2):303-7.). Upon incubation with APMA, proMMP will be activated and the resulting MMP activity (both activated MMP and already active MMP present in the sample) can be quantified.

Q: The MMP activity assay has a chromogenic read-out. Can also a fluorescent read-out be applied?

A: Yes. The kit has been developed in a chromogenic read-out because every biochemical lab has an ELISA plate reader. But if instead of the chromogenic peptide substrate a fluorescent peptide substrate is used, the read-out format will be fluorescent. Please contact QuickZyme Biosciences for more information.