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Description
Mouse NSMASE ELISA KitProduct Specification Usage Self prepared test equipment required for the experiment: 1 , plate reader ( 450nm 2 , high precision sampler and gun head: 0. 5 10uL 5 50uL 20 200uL 200 1000uL 3 37 Incubator 4 Distilled water or deionized water, Sample handling and requirements: 1 Tissue homogenate: with pre cooled PBS 0. 01M,pH=7. 4 ) Rinse the tissue to remove residual blood (lysed red blood cells in the homogenate can affect the measurement result),
Product Specification
| Usage |
Self-prepared test equipment required for the experiment: 1 , plate reader ( 450nm ） 2 , high-precision sampler and gun head: 0.5-10uL 、 5-50uL 、 20-200uL 、 200-1000uL 3 、 37℃℃ Incubator 4 Distilled water or deionized water, Sample handling and requirements: 1 Tissue homogenate: with pre-cooled PBS （ 0.01M,pH=7.4 ) Rinse the tissue to remove residual blood (lysed red blood cells in the homogenate can affect the measurement result), and break the tissue after weighing. Combine the fragmented tissue with the corresponding volume PBS (Generally according to 1 ： 9 Weight to volume ratio, such as 1g The tissue samples correspond to 9mL Of PBS The specific volume can be appropriately adjusted according to the experimental needs and recorded. Recommended in PBS Add protease inhibitor) into a glass homogenizer and ground thoroughly on ice. For further lysis of tissue cells, the homogenate can be sonicated, or freeze-thawed repeatedly. Finally, the homogenate was mixed in 5000×g Centrifugation 5~10 Minutes, take the supernatant for detection. 2 Cell lysate: pre-cooling for adherent cells PBS Gently washed, followed by trypsinization, 1000×g Centrifugation 5 Cells were collected after minutes; The suspended cells can be collected directly by centrifugation. The collected cells were pre-cooled with PBS Washing 3 Times, every 1×10^6 Added to cells 150-200uL PBS Resuspension (recommended at PBS Adding a protease inhibitor; If the content is very low, it can be appropriately reduced PBS Volume) and the cells were disrupted by repeated freeze-thaw or sonication. The extracts were mixed in 2-8℃ ， 1500×g Centrifugation 10 Minutes, take the supernatant for detection. 3 Other biological fluids: 1000xg Centrifugation 20 Minutes, take the supernatant to detect. Preparations before testing: 1 , please advance 10 Minutes remove the kit from the refrigerator and equilibrate to room temperature. 2 , Standard gradient working solution preparation: add 1mL Universal diluent into lyophilized standard and let stand 15 Minutes until it is completely dissolved and then gently mix (the concentration is 10ng/mL ） And then according to the following concentrations: 10ng/mL 、 5ng/mL 、 2.5ng/mL 、 1.25ng/mL 、 0.625ng/mL 、 0.3125ng/mL 、 0.15625ng/mL 、 0ng/mL The dilution was performed. Double dilution method : Take 7 branch EP Tube , added to each tube 500uL Universal diluent ,10ng/mL Pipette from the standard working solution 500uL To the first EP Mix evenly in a tube 5ng/mL Standard Working Solution , according to this step, absorb and mix evenly in turn. The last tube is directly used as a blank hole , there is no need to suck liquid from the penultimate tube, as shown in the figure below. 3 Preparation of biotinylated antibody detection working solution: before use 15 Min. The concentrated biotinylated antibody was concentrated in 1000×g Centrifugation 1 Minutes, in the universal diluent 100× The concentrated biotinylated antibody was diluted into 1× Working concentration (ex: 10uL Concentrate +990uL Universal Diluent) , now available for use. 4 Preparation of enzyme conjugate working solution: before use 15 Minutes will 100× Concentrated enzyme conjugate in 1000×g Centrifugation 1 Minutes, in the universal diluent 100× concentrate HRP The enzyme conjugate is diluted into 1× Working concentration (ex: 10uL Concentrate +990uL Universal Diluent) , now available for use. 5 、 1× Wash liquid preparation: Take 10mL 20× Wash liquid to 190mL In distilled water (the concentrated washing liquid taken out of the refrigerator may have crystals, which is a normal phenomenon. It can be left at room temperature and prepared after the crystals are completely dissolved). Operation steps: 1 Equilibration from room temperature 10 After minutes, remove the required slats from the aluminum foil bag, and seal the remaining slats with ziplock bag and put them back 4℃ 。 2 , Adding samples: respectively add samples or different concentration standards according to 100ul Each well is added to the corresponding well, and the blank well is added 100uL Universal diluent. After covering the sealing film 37℃ Incubation 60 Minutes. (Recommendation : Minimum dilution of sample to be tested with universal diluent 1 Times later, add the enzyme labeled plate for testing. So as to reduce the influence of matrix effect on the test results, and finally, the sample concentration needs to be multiplied by the corresponding dilution factor when calculating. It is recommended to set up double wells for all samples and standards to be tested during testing). 3 Add biotinylated antibody: take out the enzyme plate, discard the liquid without washing. Directly add biotinylated antibody working solution to each well 100uL , after covering the sealing film 37℃ Incubation 60 Minutes. 4 Plate washing: discard the liquid and add to each well 300uL 1x Wash liquid, stand 1 Minutes, throw off the washing liquid, pat dry on absorbent paper, and repeat washing the plate 3 Times (the plate can also be washed with a plate washing machine). 5 Adding enzyme conjugate working solution: adding enzyme conjugate working solution to each well 100uL , after covering the sealing film 37℃ Incubation 30 Minutes. 6 Plate washing: discard the liquid according to the steps 4 Washing method, wash plate 5 Times. 7 Substrate addition: substrate is added per well ( TMB ） 90uL Covered with a sealing film, 37℃ Incubation protected from light 15 Minutes. 8 Add stop solution: take out the enzyme label plate and directly add stop solution to each well 50uL , immediately in 450nm Wavelength measurement of each well OD Value. Calculation of experimental results: 1 Result judgment: 1 , calculate the average of the standard and sample replica well OD Value and subtract the blank hole's OD Values as correction values. Taking concentration as the abscissa, OD Value is ordinate , draw the standard curve of the four-parameter logic function on the double logarithmic coordinate paper. 2 If the sample OD If the value is higher than the upper limit of the standard curve, the test should be retested after appropriate dilution and multiplied by the corresponding dilution factor when calculating the sample concentration. |
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| Theory | This kit uses double antibody sandwich enzyme-linked immunosorbent assay (ELISA). To the microwells pre-coated with Sphingomyelin phosphodiesterase 2 (NSMASE) capture antibody, sample, standard, biotin-labeled detection antibody, and HRP enzyme conjugate were added sequentially, incubated and washed in the middle, and colored with the substrate TMB. TMB is converted to blue under the catalysis of peroxidase (HRP) and to the final yellow under the action of acid. There was a positive correlation between the depth of color and Sphingomyelin phosphodiesterase 2 (NSMASE) in the sample. The absorbance (0D value) was measured with a microplate reader at a wavelength of 450nm, and the sample concentration was calculated. | |||||||||||||||||||||||||||||||||
| Source | Mouse | |||||||||||||||||||||||||||||||||
| Synonym | Mouse Sphingomyelin phosphodiesterase 2 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Neutral sphingomyelinase (NSMASE), also known as sphingomyelin phosphodiesterase 2 (SMPD2), is an enzyme encoded by the SMPD2 gene. The gene encodes a protein that was initially identified as sphingomyelinase based on sequence similarity between bacterial sphingomyelinase and yeast proteins. Subsequent studies have shown that its biological function is unlikely to be as a sphingomyelinase, but rather as a lysophospholipase. | |||||||||||||||||||||||||||||||||
| General Notes | 1. Carry out incubation in strict accordance with the specified time and temperature to ensure accurate results. All reagents must reach room temperature 20-25 °C prior to use. Store reagents in refrigeration immediately after use. 2. Incorrect plate washing may lead to inaccurate results. Make sure to drain the liquid from the wells as much as possible before adding the substrate. Do not allow the wells to dry out during incubation. 3. Eliminate the residual liquid and fingerprints at the bottom of the plate, otherwise it will affect the OD value. 4. The substrate color development solution should be colorless or very light in color, and the substrate solution that has turned blue cannot be used. 5. Avoid cross-contamination of reagents and specimens to avoid wrong results. 6. Avoid direct exposure to strong light during storage and incubation. 7. Any reaction reagent cannot come into contact with the bleaching solvent or the strong gas emitted by the bleaching solvent. Any bleaching component will destroy the biological activity of the reaction reagents in the kit. 8. Expired products cannot be used, and components with different item numbers and batch numbers cannot be mixed. 9. Recombinant proteins from sources other than the kit may not match the antibodies in this kit and are not recognized. 10. If the disease may be spread, all samples should be managed well, and the samples and testing devices should be handled according to the prescribed procedures. |
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| Storage Temp. | Unopened kit, stored at 4 °C, shelf life 6 months. | |||||||||||||||||||||||||||||||||
| Test Range | 0.156-10ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates and other biological fluids |
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4.9 ★★★★★
Based on 14 reviews
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Product Reviews
★★★★★ 3
Good-ish
Format: Kindle
🌶️ 0/5
⭐️ 3/5
I will preface this by saying I am not a huge RH fan. However it didn’t feel like a RH quite yet. The relationships and plot are all building in this book. At first I was thinking things were happening stupid fast since she’s had zero interaction with another being and has been tortured her whole life, and I believe they were for one side of the relationship, but by 50% things moved too slow lol. Idk I think the main 1-3 guys I’m actually interested in more than the others didn’t get enough air time so I hope the next book things start moving with them since it ended the way it did.
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Reviewed in the United States on April 12, 2025
★★★★★ 5
Can I have Term 2 now?
Format: Kindle
This is by far my favorite book of Lyra's to date. I gobbled this book up so fast, I was so upset I finished it. I know this is going to be a book I reread, just like Fate Hollow Academy.
I loved Fate Hollow Academy, so I was excited when Lyra announced she was taking us readers back to Kalista. Although the characters from Fate Hollow are mentioned several times throughout the book, this book is about Pandora and her love interests in the Demon Realm.
Before you deep dive into this book, definitely look at the trigger warnings before you start. When Lyra says there's dark themes in this book, she meant it. Also, just know this book does end on a cliffhanger and will probably leave you with as many questions as it did me.
Below may contain some minor spoilers, so keep that in mind if you want to keep reading my review.
Pandora's first 2 decades of her life are full of torture, hatred, and confinement in a cellar. That is until her father finds her and brings her into the world she was supposed to live in. Because of her upbringing she wasn't acclimated to the Demon world and the way their society works so when she is offered the chance to go to a Demon Reform Accademy, she accepts it to learn the ways of society and how she is supposed to feed.
Pandora isn't like the other common demons in this world, no. She, like her father Death, are soul-eaters. Pandora is too scared to release her powers because she doesn't want to kill anyone, so she needs to learn how to feed off of a piece of soul instead of taking the whole thing.
Because of who her father is, she's classed as a Noble, and while she doesn't agree with her fellow Noble students, who look down on the "lower" class, she also is faced with 3 Demons who hate her for being a Noble. Theses a lot of back and forth tension which these 3 demons who have issues of their own. One is obsessed with her, and wants to know all of her secrets. One leaks fear all over the place, but also is scared of the "princess" -wink- -wink-. One is a complete drunk who doesn't like her just because he's got family Noble problems.
Don't worry, there are 2 other demons who are just the sweetest towards her. One who will get vengeance for anything done to her, he's got Golden Retriever energy around her and touch her and die energy for anyone around her. Finally, there is one who is friends with her even when the whole school is scared of her, who brings her into his dreams and will create nightmares for those who bother her.
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Reviewed in the United States on June 4, 2024
★★★★★ 4
This was more than I expected...
Format: Kindle
So...I finished this book at nearly 2am! It was just that hard to put down. I loved that it's set in the same world as Fates Hollow but in the future. Poor Pandora has been through so much but there's light at the end of the tunnel for her. She finally meets her dad and it turns out he's amazing. In order to help her learn about demon society and how to control her magic he sends her to the Demon Reform Academy. There she meets 2 awesome demons who help her and care for her. She also sadly meets her 3 tormentors. I will say though that while they do bully Pandora they also rescue her several times and defend her. They too have traumatizing pasts and can't see the good that is in front of them. Of course demon society needs to be shaken up too since that's part of the problem. I really enjoyed this read. I teared up a few times throughout the book but I liked the characters and that ending...well it looks like 3 in denial are going to need to grovel hard.
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Reviewed in the United States on June 1, 2024
★★★★★ 5
MUST READ BOOK!!
Format: Kindle
I was in a huge reading slump, when one of my favorite authors recommended this book in her readers group (The amazing Marie Mistry 🫶) and I absolutely DEVOURED this book. Now I’m in the club crying about having to wait until December for Term 2!
This book is about a girl named Pandora who has suffered a life that nobody would ever wish to have. This book is about Pandora learning how to live, learning about herself and what she really wants. She has a long journey to find out all of these things, but she took the first steps in this book and it was beautiful to read. Some of the men in this book are our dream men, Hunter and Reed 😍 and then we have the men who need some work and who we all really just want to beat up until they admit what they really feel, Skel, Bram and Dexter. But that cliffy was a killer and I absolutely cannot wait until the next book!!
You have written an absolute dream of a book Lyra and I eagerly await the next installment in this series!!!
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Reviewed in the United States on June 7, 2024
★★★★★ 5
OUTSTANDING
Format: Kindle
This was very, very good. The world is vast and characters are complex. There is a good plot with a whole lot going on. This is well written. Good twists and turns and some heart breaking moments. You will love these characters, they have heart and loyalty. I am hoping that there will be several more books. We've yet to see anything from the Sea Court but only a mention of them here and there. The Wood Court was given a quick couple of scenes, and only as far as some warriors, we've yet to enter their court and the Shadow Court, I'm not sure if they will be a force for good or bad, but they definitely will play a much bigger role moving forward. This is primarily the Ice and Air Courts. Told in multiple views, which I loved, it gives you a chance to see things from different eyes. There's alot of political maneuvering and deception. I loved it and will pick up the next book as it becomes available. If you like The Fae and the courts, you should love this. I think the author has mucn in store for us.
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Reviewed in the United States on March 8, 2020