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KB00001 Samples from which species can be used with the PPAR Complete Transcription Factor Assay? Human and mouse samples are suitable for use with this kit according to the kit booklet instructions. While the kit has not been specifically validated with rat samples, we expect that there should be sufficient homology between the rat and humans sequence for both the antibody and dsDNA in the kit to allow detection of PPARs from samples of rat origin. PPARα, δ, γ Complete Transcription Factor Assay Kit
KB00002 In the Ghrelin (human acylated) EIA Kit when using plasma samples, is it possible to use AEBSF rather than PHMBA? It appears that the AEBSF interferes with the enzyme activity of the acetylcholine esterase on the tracer in the Ghrelin (human acylated) EIA Kit. There is a way around this. It is possible to perform the assay in a two step process. The first step is incubating samples and standards with the immobilized antibody on the plate. The second step involves washing the wells to remove the protease inhibitors then incubation with the detection antibody/AChE conjugate. Ghrelin (human acylated) EIA Kit
KB00003 Is the Glutathione S-Transferase Polyclonal FITC Antibody a FITC anti-glutathione S-transferase antibody that is suitable for flow cytometry? Yes, this product contains fluorescein that is conjugated to an anti-glutathione S-transferase antibody which is useful for flow cytometry. Glutathione S-Transferase Polyclonal FITC Antibody
KB00007 What are the main differences between the Nitrate/Nitrite Colorimetric Assay Kit and Nitrate/Nitrite Colorimetric Assay Kit (LDH method)? The Nitrate/Nitrite Colorimetric Assay , product number 780001, uses a NADPH cofactor recycling method which allows use of a NADPH concentration that is below the level of interference in the Griess reaction. The Nitrate/Nitrite Colorimetric Assay (LDH Method), product number 790871 uses LDH to eliminate all excess NADPH that is required for NOS activity assays. The LDH kit also removes excess NADPH used to catalyze conversion of Nitrate to Nitrite. Of the two nitrate/nitrite assays mentioned, we recommend the Nitrate/Nitrite Colorimetric Assay (or the corresponding Fluorometric assay if greater sensitivity is needed) for most applications since it is significantly easier to perform. The only reason to use the Nitrate/Nitrite Colorimetric Assay Kit (LDH method) is for measurement of Nitrate/nitrite from in vitro NOS activity assays. Nitrate/Nitrite Colorimetric Assay Kit
KB00008 What are the main differences between the Nitrate/Nitrite Colorimetric Assay Kit and Nitrate/Nitrite Colorimetric Assay Kit (LDH method)? The Nitrate/Nitrite Colorimetric Assay , product number 780001, uses a NADPH cofactor recycling method which allows use of a NADPH concentration that is below the level of interference in the Griess reaction. The Nitrate/Nitrite Colorimetric Assay (LDH Method), product number 790871 uses LDH to eliminate all excess NADPH that is required for NOS activity assays. The LDH kit also removes excess NADPH used to catalyze conversion of Nitrate to Nitrite. Of the two nitrate/nitrite assays mentioned, we recommend the Nitrate/Nitrite Colorimetric Assay (or the corresponding Fluorometric assay if greater sensitivity is needed) for most applications since it is significantly easier to perform. The only reason to use the Nitrate/Nitrite Colorimetric Assay Kit (LDH method) is for measurement of Nitrate/nitrite from in vitro NOS activity assays. Nitrate/Nitrite Colorimetric Assay Kit (LDH method)
KB00009 What is the main difference between 8-Isoprostane EIA Kit and 8-Isoprostane Express EIA Kit? Between the two 8-Isoprostane EIA kits you mention, 8-Isoprostane EIA Kit requires an overnight incubation and has a detection limit of 2.7 pg/ml. The 8-Isoprostane Express EIA Kit, can be performed in one day but has a detection limit of 10 pg/ml. 8-Isoprostane EIA Kit
KB00012 What is the recommended concentration of nuclear protein that should be added to each well for Transcription Factor Assay Kits? For the Transcription Factor Assay Kits, 10 µg of nuclear protein per 10 µl is recommended. Transcription Factor Assay
KB00015 In the various Transcription Factor Assay Kits, can whole cell lysates be used in place of nuclear extracts? We recommend using nuclear extracts for transcription factor assay kits. The effective use of whole cell lysate in the assay will depend on the cell type and the activation state of the cells. Transcription Factor Assay
KB00016 Have the Transcription Factor Assays been validated for use with tissue homogenates? The Transcription Factor Assays have not been validated with tissue samples in our labs. However, we have reports from customers who have had success with tissue samples. Transcription Factor Assay
KB00017 Can Transcription Factor Assays be used to assess transcriptional activity? Transcription Factor Assays do not measure transcription activity. The assay measures specific transcription factor DNA binding activity. For example,in the PPAR Transcription factor assays, PPPARs present in the sample will bind to the PPRE and there is no reporter indicating the activity of the PPAR. There have been no studies done to examine whether PPAR bound to the PPRE is active or not. Transcription Factor Assay
KB00019 What is the detection limit in the PPAR Complete Transcription Factor Assay Kit? The transcription factor assays are not quantitative in nature but rather rely on comparison of absorbance values relative to various sample treatments. They are also highly dependent on the cell type and nature of the cell stimulation. For example, we start to see an absorbance above background at about 1-2 µg of activated 3T3L1 cells. PPARα, δ, γ Complete Transcription Factor Assay Kit
KB00021 In the Transcription Factor Assays how much nuclear protein should be loaded into each well? The kit booklet protocol starts with 10,000,000 cells per condition. This will result in approximately 50 µg of protein in 50 µl of nuclear extract, or ~10 µg per well in 10 µl of nuclear protein. Transcription Factor Assay
KB00022 In the Transcription Factor Assays how do you report the results? The Transcription Factor Assays do not have a standard curve. The endpoint is the absorbance at 450 nm. There is a positive control in the assay, but this is only intended as a qualitative assessment of assay performance, not as a quantitative standard. The experimental design should include a control with which to compare treatment groups. Transcription factor binding can be reported as a percentage of control or a fold increase. Transcription Factor Assay
KB00023 Can an IC50 be calculated for the PPAR Complete Transcription Factor Assay Kit? Yes, even though there is no standard curve in the assay, IC50's of receptor antagonists can be calculated. In this case, the receptor must first be activated to 100% by the natural ligand. The IC50 would then be determined by a finding the concentration of antagonist that causes a reduction of signal by 50 percent. Transcription Factor Assay
KB00024 Are the Transcription Factor Assays compatible with other commercial nuclear extraction kits? We are not sure of the compatibility with all other commercial nuclear extraction kits. The Transcription Factor Assays are sensitive to the salt concentration of the buffer used for the nuclear extract. It is preferable to use the protocol in our kit booklet, or we offer a Nuclear Extraction Kit (Item No. 10009277). Transcription Factor Assay
KB00026 In the PPAR Complete Transcription Factor Assay Kit, are the PPAR antibodies specific only to each subtype? The antibodies are specific for each specific subtype. PPARα, δ, γ Complete Transcription Factor Assay Kit
KB00027 In the S-Nitrosylated Protein Detection Kit is there any point early in the procedure where the protocol can be stopped overnight? It is possible to stop the procedure at either of the two acetone protein precipitations (step 7 or step 13 under the Sample Preparation section). At either point you can leave the precipitates at -20°C overnight. S-Nitrosylated Protein Detection Kit
KB00028 If no dark room is available, how important is the performance of steps 1-12 under indirect light in the S-Nitrosylated Protein Detection Kit protocol? If you can turn off some of the overhead lights in the lab and avoid using a lamp directly on the bench, that will help. S-Nitrosylated Protein Detection Kit
KB00030 In the 8-isoprostane EIA Kit, what amount of fresh homogenized tissue is needed? There is no fixed amount of tissue required for the assay, as the amount of tissue needed will vary depending on the experimental conditions. Sample purification for tissues is recommended as described in the kit booklet. 8-Isoprostane EIA Kit
KB00031 What is the procedure for running a cross reactivity check in a competitive EIA kit like the Prostaglandin EIA Kit - Monoclonal? To test cross-reactivity in our EIA kits, use the compound of interest as a standard, and compare the IC50 of the cross reactive compound to the IC50 of the standard. We generally start at 10,000 x the concentration of S1 so that we can determine cross reactivity of greater than or equal to 0.01%. On the plate set up NSBs, B0s, the regular standard curve, and a standard curve of the cross-reacting compound. The fold dilution of the cross-reacting compound will depend on the starting concentration. We usually do ten-fold dilutions with compounds where we want to test a really broad range of concentrations. We change pipet tips between dilutions – with ten-fold dilutions the carry-over effect is enough to throw off cross-reactivity determinations. After performing the assay as normal, divide the IC50 of the standard by the IC50 of the cross-reactive compound and multiply by 100 to get percent cross reactivity. For example, if the IC50 is 50 pg/ml and the IC50 of the cross reactive compound is 5 ng/ml, cross reactivity = ((50 pg/ml)/(5000 pg/ml))*100 = 1% Prostaglandin E2 EIA Kit - Monoclonal
KB00032 For the Protein Carbonyl Assay Kit are there recommendations for use of samples dissolved in buffer that is 30 mM Tris-HCl, 7 M Urea, 2 M Thiourea, 4% CHAPS, Protease inhibitor cocktail, pH 8.5? The buffer you are using is designed to solubilize proteins. It is possible that the protein in your samples will have a difficult time precipitating in the assay. The carbonyl groups in urea and CHAPS should not react with the DNPH in the assay, but it is unknown whether or not these components will interfere (as other substances that contain no carbonyl groups can still interfere, such as Triton X-100). The assay may suit your needs but we cannot guarantee this. Protein Carbonyl Assay Kit
KB00033 With what species is the Vasopressin EIA kit compatible? Vasopressin has the same nonapeptide sequence across mammalian species so it should work well for all mammalian samples. Arginine Vasopressin EIA Kit
KB00034 What is the difference between each of the mouse anti-rabbit IgG coated plates (associated with the catalog numbers 400004, 400005, 400006, and 400007)? The plates for Item numbers 400004 and 400005 consist of 12 detachable strips containing 8 wells each. These plates are coated with mouse monoclonal anti-rabbit IgG in which the coating antibody originates from different clones. Items 400006 and 400007 are the corresponding solid plate versions with the same antibody coating as 400004 and 400005, respectively. Precoated (Mouse Anti-Rabbit IgG) EIA 96-Well Strip Plate
KB00037 On the product insert for Arachidonoyl Ethanolamide-d8 it states the chemical purity is 97% and contains no d0 (undeuterated compound). Are there other deuterated forms present? In our catalog we state that the Arachidonoyl Ethanolamide-d8 is less than or equal to 1% of the undeuterated compound. There can be other deuterated forms. We can assure you that the compound does have less than 1% of the undeuterated compound present. Arachidonoyl Ethanolamide-d8
KB00038 For the 11-dehydro Thromboxane B2 EIA Kit with urine samples and purfication with SPE cartridges how is the 63 mM ammonium bicarbonate and the 0.63 mM ammonium bicarbonate prepared? To prepare the ammonium bicarbonate buffers you may do the following: for one liter of 63 mM ammonium bicarbonate buffer, add 4.98 g NH4HCO3 in about 800 ml of purified water in a beaker with a magnetic stir bar and adjust the pH with concentrated HCl until pH 8.6; then transfer to a graduated cylinder and add water to one liter. For the 0.63 mM ammonium bicarbonate buffer, you may use the same procedure except add 0.05 g of ammonium bicarbonate.
KB00041 Are serum samples suitable for Lipid Hydroperoxide (LPO) Assay Kit? Yes, serum samples may be suitable for the LPO Assay Kit. In the kit booklet there is a procedure in the Sample Preparation for plasma which is suitable for serum also. Lipid Hydroperoxide (LPO) Assay Kit (96 well)
KB00042 Are serum samples suitable for the Lipid Hydroperoxide (LPO) Assay Kit ? Yes, Serum samples are fine for the LPO Assay Kit. In the kit booklet there is a procedure in the Sample Preparation for plasma which is suitable for serum also. Lipid Hydroperoxide (LPO) Assay Kit
KB00043 Are 8-Isoprostane and 8-iso-Prostalgandin F2α the same thing? Yes, 8-Isoprostane and 8-iso-Prostaglandin F2α are synonyms. 8-Isoprostane EIA Kit
KB00044 Can the Glutathione S-Transferase Assay Kit kit be used for porcine samples? This kit has not been tested specifically with porcine samples, but the assay should work with all GSTs. Since GSTs are highly conserved enzymes, this kit should work well for samples from any species. Glutathione S-Transferase Assay Kit
KB00045 Is the EP4 Receptor (N-Term) Polyclonal Antiserum purified? No. This product is referred to as antiserum because the product is actually rabbit serum that has not been purified. Although antibodies supplied as antiserum often give a superior signal on western blots, you should be aware that the antiserum preparation may also produce additional bands compared to an affinity-purified version of the antibody. EP4 Receptor (N-Term) Polyclonal Antiserum
KB00046 What is the source of Cayman's Prostaglandin E2? Cayman's Prostaglandin E2 is synthesized. Prostaglandin E2
KB00047 What is recommended for IP injections of FTY720 in mice and are there any references? For IP injections in mice, it is recommended to use the parent compound and not the phosphates or azido compounds. A Google Scholar search for "FTY720 in vivo" returned over 3,000 matches. From the first few articles it appears that FTY720 can be dissolved in various aqueous solutions including water, culture media, normal saline, and H2O:Me2SO (5:1 v/v). Some of these studies used oral gavage and IP injection. FTY720
KB00048 For the Protein Carbonyl Assay Kit You indicate the following equation; Protein Carbonyl(nmol/ml)=[(CA)/(*0.011uM^-1)](500ul/200ul) Does an actual calculation simply divide CA by 0.011? What do you mean "(500 µl/200 µl)" ? Dividing CA by 0.011µM^-1 will give the concentration of protein carbonyl for the 500µl guanidine hydrochloride solution. Performing the 500µl/200µl will give the concentration of protein carbonyl in the original 200µl sample. If a customer only used a 100µl sample, for example, the equation would be adjusted to 500µl/100µl. This term in the equation accounts for sample dilution. Protein Carbonyl Assay Kit
KB00049 In the S-Nitrosylated Protein Detection Kit there is not a protocol for use of a whole tissue homogenate. There is a protocol for isolated tissue cell pellet, but nothing starting from whole tissues. Is there a way to use this kit? For a soft tissue where cells can be isolated one may: isolate, wash and pellet the cells, pick up at step 3 to lyse cells and block free thiols. For solid tissue samples, homogenize at 0.1 g/ml buffer in 20 mM Tris pH 8.0, or P-SNO Cell Lysis Buffer (Buffer A). Centrifuge here to eliminate major debris (1000xg, 10 minutes). Repeat the homogenization with the ice-cold supernatant and sonicate if needed (to ensure complete lysis, check for remaining intact cells by microscopy). Quantify the protein concentration and dilute to 2 mg/ml if needed. Treat the sample going forward as a cell lysate by adding the reconstituted blocking reagent (1 mL stock solution) to the current sample volume in 1:9 proportion. S-Nitrosylated Protein Detection Kit
KB00050 The the level of 8-OH-dG has been estimated in cultured cells after extracting total DNA. What is the best way to normalize the data for accurate comparison between treatments? It is more meaningful to normalize measured values to the total number of cells, or the amount of purified DNA rather than as a concentration of 8-OH-dG only. 8-hydroxy-2-deoxy Guanosine EIA Kit
KB00051 Has the 8-hydroxy-2-deoxy Guanosine EIA Kit been validated for muscle tissue or whole blood (leukocyte DNA) samples? The 8-hydroxy-2-deoxy Guanosine EIA Kit has not been validated for those exact sample types, but has been validated for cell lysates and tissue homogenates in general. Theoretically, the kit should work just fine with any purified DNA sample. 8-hydroxy-2-deoxy Guanosine EIA Kit
KB00052 Is the Angiotensin II EIA Kit suitable for use with rat plasma and supernatant of cell culture? The sequence for angiotensin II is conserved across mammalian species. Therefore, this kit will work with rat samples, however purification is required. Angiotensin II EIA Kit
KB00053 What is the solubility of Xestospongin in water? Xestospongin is only slightly soluble in aqueous solvents. It is suggested to first dissolve the compound in an organic solvent and then make dilutions with an aqueous solvent to get it into any type of aqueous solution. Xestospongin C
KB00054 What is the effect of BSA in samples in the Prostaglandin E2 EIA Kit - Monoclonal? The EIA buffer, the medium that the Prostaglandin E2 EIA Kit - Monoclonal is performed in, contains 0.1% BSA as its working concentration. Also samples are diluted in this buffer. Unless you are planning on using significantly higher levels of BSA, your samples should be okay. Assaying all samples at two dilutions is recommended. Prostaglandin E2 EIA Kit - Monoclonal
KB00055 Is there an optimized sample concentration or tissue weight for the Lipid Hydroperoxide (LPO) Assay Kit ? In the Lipid Hydroperoxide (LPO) Assay Kit booklet you are directed to use a known amount of sample and an equal volume of Extract R saturated methanol. Unfortunately we do not have values for quantities of tissue samples to suggest. There are so many tissue types that it would be difficult to suggest quantities. We suggest that in the course of your experiment you try a few different quantities and dilutions to optimize the concentration. Lipid Hydroperoxide (LPO) Assay Kit
KB00056 In the COX Activity Assay Kit, what is the principle of the reaction of TMPD by peroxidase? In the Background section of the COX Activity Assay Kit booklet, it states that the COX component converts arachidonic acid to Prostaglandin G2 (PGG2) and the peroxidase component reduces the PGG2 to the corresponding alcohol, PGH2. In this kit the TMPD serves as a reducing agent, TMPD gets oxidized, so electrons flow from the TMPD to PGG2 and the appearance of the oxidized. TMPD is monitored at 590 nm. COX Activity Assay Kit
KB00057 What is the density of BADGE? The density of BADGE is 1.17 g/ml. BADGE
KB00058 What is the concentration for the Synthetic Cannabinoid HPLC Mixture? Thie Synthetic Cannabinoid HPLC Mixture is made by combining 100 µl of a 1 mg/ml stock of each compound for at total volume of 1.2 ml (final concentration of 83.3 µg/ml for each compound). Synthetic Cannabinoid HPLC Mixture I
KB00059 Does the iPLA2 (type VI) Polyclonal Antiserum recognize the γ or the β form? The sequence analysis suggests the iPLA2 (type VI) Polyclonal Antiserum only recognizes the β isoforms (2 isoforms: short = 752 and long = 806 amino acids) and does not recognize iPLA2γ. iPLA2 (Type VI) Polyclonal Antibody
KB00060 Does the PPAR Complete Transcription Factor Assay Kit measure all three isoforms of PPARα, δ, and γ ? Yes, the PPAR Complete Transcription Factor Assay Kit can measure all three isoforms of PPARα, δ, and γ. There are enough reagents for one third of a plate for each isoform. PPARα, δ, γ Complete Transcription Factor Assay Kit
KB00061 What cell types have been used with the PPAR Complete Transcription Factor Assay Kit? We have used the PPAR Complete Transcription Factor Assay Kit successfully with 3T3-L1 cells and HepG2 cells. Many other cells types may be applicable depending on their function and levels of individual PPAR isoforms present. PPARα, δ, γ Complete Transcription Factor Assay Kit
KB00063 Can the Glutathione S-Transferase Polyclonal FITC Antibody be used for flow cytometry? For the Glutathione S-Transferase Polyclonal FITC Antibody, an application in flow cytometry has not been investigated. The intended use of the antibody is for monitoring S. japonicum GST-tagged recombinant proteins. Glutathione S-Transferase Polyclonal FITC Antibody
KB00065 When ready to develop a plate in a EIA kit, the protocol says to empty the wells and rinse with the wash buffer five times. How should the wells be emptied? The plate may be emptied into the sink and then tapped onto paper towels to remove residual wash buffer. EIA (Enzyme Immunoassay)
KB00066 If the level of LPO in the samples is low how is this handeled with Lipid Hydroperoxide (LPO) Assay Kit? With the Lipid Hydroperoxide (LPO) Assay Kit there is no procedure for concentrating the sample. When the level of lipid peroxides in a sample is below the detection limit of the LPO Assay, and a more sensitive assay for lipid oxidation is required. In these cases we suggest using the 8-Isoprostane EIA Kit, item # 516351, which assess the levels of a stable end product of lipid oxidation. Lipid Hydroperoxide (LPO) Assay Kit
KB00067 Is there a protocol for serum sample preparation for the Lipid Hydroperoxide (LPO) Assay Kit? In the Lipid Hydroperoxide (LPO) Assay Kit booklet there is a procedure for sample preparation for plasma which is suitable for serum also. Lipid Hydroperoxide (LPO) Assay Kit (96 well)
KB00069 What type of plasma can be used in the Glutathione S-Transferase Assay Kit? Any of the anticoagulants used in plasma such as heparin, citrate, or EDTA are suitable for the Glutathione S-Transferase Assay Kit. Glutathione S-Transferase Assay Kit
KB00070 What is the stability of a stock solution of Prostaglandin E2, prepared by dissolving the crystalline compound in an organic solvent such as ethanol? The two year stability listed is only for the crystalline solid, not for a solution in an organic solvent. As solution in an organic solvent Prostaglandin E2 stored at - 20°C is likely to be good for several weeks. Prostaglandin E2
KB00071 Is purification needed for plasma samples prior to use in the Angiotensin II EIA Kit? Yes, plasma samples need purification prior to use in the Angiotensin II EIA Kit. The chemicals are available from Sigma: o-Phenanthroline Catalog number 33510, p-hydroxymercuribenzoic acid Catalog number 55540 Pepstatin A Catalog number 77170. The phenyl cartridge is from Phenomenex/Strata. The catalog numbers are: 1 ml Cat 8B S006 EAK , 3 ml Cat 8B S006 HBJ. Angiotensin II EIA Kit
KB00073 For the S-Nitrosylated Protein Detection Assay Kit is it is okay to have β-mercaptoethanol in the Laemmli buffer? Yes, it is okay to have β-mercaptoethanol in the Laemmli buffer in the S-Nitrosylated Protein Detection Assay Kit. The Biotin will not dissociate from the protein. S-Nitrosylated Protein Detection Kit
KB00076 Is there a protocol for analysis with the Bio-active Lipid I Screening Library? Cayman provides this grouping of compounds to facilitate their use in high throughput screening projects determined by the individual investigators. No, a protocol for analysis is not currently provided. Bio-active Lipid I Screening Library (96-Well)
KB00077 Has progesterone 3-biotin has ever been tested for interaction with progesterone receptor? In other words, can the progesterone 3-biotin be used for progesterone receptor pull-down? We have no data on the interaction of progesterone-3-biotin with the progesterone receptor. Since steroid structure is often maintained at or near the 3 position, there may be a good chance this portion does not interact significantly with the receptor. This may imply that the biotin attachment may not interfere with receptor binding. Progesterone 3-biotin
KB00078 Can the Mouse anti-striped bass Vtg Monoclonal antibody, ND-3G2, product number 170107, be used with common Mummichog Fundulus heteroclitus? We have not tested our antibody against Mummichog so we don´t know for sure. However, we have tested product 170107 against Vtg from two other fish from the order Cyprinodontiformes (Killifish and Sheepshead minnow) and the antibody cross-reacts with both of them so there is a good chance it may work. Vitellogenin (striped bass) Monoclonal Antibody (ND-3G2)
KB00079 Can the Rainbow trout vitellogenin standard, product number 470164, be used with common Mummichog Fundulus heteroclitus? No, due to the variation in structure of Vtg from different species, unfortunately you cannot use a standard from one species with another fish. Vitellogenin (rainbow trout) Standard
KB00080 What sample types can be used with the S-Nitrosylation Protein Detection Kit? The protocol in the S-Nitrosylated Protein Detection Kit (cat. 10006518) refers to use of a cell pellet which can be obtained from cultured cells, or blood cells. Alternatively, the assay can be used with purified protein. If using a purified protein, begin by resuspending the protein sample and incubating at 2mg/mL in Buffer A containing Blocking Reagent; this will keep the blocking reagent in excess. At this point, free thiols on the protein are blocked and you can continue with the acetone precipitations in step 6. For gel analysis, load only 1-5 µg of protein per lane to avoid overloading the protein. S-Nitrosylated Protein Detection Kit
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