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Description
Human E-selectin ELISA KitProduct Specification Usage Self prepared test equipment required for the experiment: 1. Microplate reader (450nm) 2. High precision sampler and gun tip: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37 incubator 4. Distilled water or deionized water Sample handling and requirements: Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or 4 overnight, then centrifuge at 1000 g for 20 minutes, take the
Product Specification
| Usage |
Self-prepared test equipment required for the experiment: 1. Microplate reader (450nm) 2. High-precision sampler and gun tip: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37 ℃ incubator 4. Distilled water or deionized water Sample handling and requirements: Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or 4 ℃ overnight, then centrifuge at 1000 × g for 20 minutes, take the supernatant, or store the supernatant at-20 ℃ or-80 ℃, but avoid repeated freezing and thawing. Plasma: Collect specimens with EDTA or heparin as anticoagulants, and centrifuge the specimens at 2-8 ℃ 1000 × g for 15 minutes within 30 minutes after collection. Take the supernatant for detection, or place the supernatant at-20 ℃ or-80 ℃ for storage, but avoid repeated freezing and thawing. Tissue homogenate: Wash the tissue with pre-cooled PBS (0.01 M, pH = 7.4) to remove residual blood (lysed red blood cells in the homogenate will affect the measurement results), and break the tissue into pieces after weighing. Combine the crushed tissue with the corresponding volume of PBS (generally according to the weight-to-volume ratio of 1: 9, for example, 1g of tissue sample corresponds to 9mL of PBS. The specific volume can be appropriately adjusted according to the needs of the experiment and recorded. It is recommended to add protease inhibitor to PBS) add to a glass homogenizer and grind thoroughly on ice. For further lysis of tissue cells, the homogenate can be sonicated, or freeze-thawed repeatedly. Finally, the homogenate was centrifuged at 5000 × g for 5-10 minutes, and the supernatant was taken for detection. Cell lysate: Adherent cells were gently washed with pre-cooled PBS, then digested with trypsin, centrifuged at 1000 × g for 5 minutes and collected; The suspended cells can be collected directly by centrifugation. The collected cells were washed 3 times with pre-cooled PBS, and 150-200uL PBS was added to every 1 × 10 ^ 6 cells to resuspend (it is recommended to add protease inhibitors to PBS; if the content is very low, the PBS volume can be appropriately reduced) and the cells were disrupted by repeated freezing and thawing or ultrasound. The extract was centrifuged at 2-8 °C at 1500 × g for 10 minutes, and the supernatant was taken for detection. Cell culture supernatant: Please centrifuge at 1000 × g for 20 minutes, take the supernatant for detection, or store the supernatant at-20 ℃ or-80 ℃, but avoid repeated freezing and thawing. Other biological liquids: centrifuge at 1000xg for 20 minutes, and take the supernatant for detection. Preparations before testing: 1. Please take out the kit from the refrigerator 10 minutes in advance and balance it to room temperature. 2. Preparation of standard gradient working solution: Add 1mL of universal diluent to the freeze-dried standard, let it stand for 15 minutes until it is completely dissolved, and then gently mix it (the concentration is 10ng/mL), and then dilute according to the following concentrations: 10ng/mL, 5ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, 0.15625 ng/mL, 0ng/mL. Double dilution method: Take 7 EP tubes, add 500uL of universal diluent to each tube, draw 500uL of 10ng/mL standard working solution into the first EP tube and mix well to prepare 5ng/mL standard working solution. According to this step, suck and mix well in sequence. The last tube is directly used as a blank hole, so 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: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000 × g for 1 minute, and dilute 100 × concentrated biotinylated antibody to 1 × working concentration with universal diluent (example: 10uL concentrated solution + 990uL universal diluent), now prepared for use. 4. Preparation of enzyme conjugate working solution: Centrifuge 100 × concentrated enzyme conjugate at 1000 × g for 1 minute 15 minutes before use, and dilute 100 × concentrated HRP enzyme conjugate to 1 × working concentration with universal diluent (example: 10uL concentrated solution + 990uL universal diluent), now prepared for use. 5. Preparation of 1 × washing liquid: Take 10mL of 20 × washing liquid into 190mL distilled water (the concentrated washing liquid taken out of the refrigerator may have crystals, which is a normal phenomenon. It can be placed at room temperature and then prepared after the crystals are completely dissolved). Operation steps: 1. Take out the required slats from the aluminum foil bag after equilibration at room temperature for 10 minutes, and seal the remaining slats with a ziplock bag and put them back to 4 °C. 2. Add samples: Add samples or standards of different concentrations to the corresponding wells at 100uL per well, and add 100uL of universal diluent to the blank wells. Incubate at 37 °C for 60 minutes after covering the plate sealing film. (Recommendation: Dilute the sample to be tested with a universal diluent at least 1 times and then add it to the enzyme labeled plate for testing. This reduces the influence of matrix effect on the test results. 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 labeled plate, discard the liquid, and do not wash it. 100 uL of biotinylated antibody working solution was directly added to each well, and the plate sealing membrane was covered and incubated at 37 °C for 60 minutes. 4. Plate washing: Discard the liquid, add 300uL 1x washing liquid to each hole, let it stand for 1 minute, throw away the washing liquid, pat dry on absorbent paper, and repeat washing the plate 3 times (you can also use a plate washing machine to wash the plate). 5. Add enzyme conjugate working solution: Add 100uL of enzyme conjugate working solution to each well, cover the sealing membrane and incubate at 37 °C for 30 minutes. 6. Wash the plate: Discard the liquid and wash the plate 5 times according to the washing method in step 4. 7. Add substrate: Add 90uL of substrate (TMB) to each well, cover with a sealing film, and incubate at 37 °C in the dark for 15 minutes. 8. Add stop solution: Take out the enzyme plate, directly add 50uL of stop solution to each well, and immediately measure the OD value of each well at a wavelength of 450nm. Calculation of experimental results: Result judgment: 1. Calculate the average OD value of the standard product and the sample double well and subtract the OD value of the blank well as the correction value. Taking concentration as abscissa and OD value as ordinate, the standard curve of four-parameter logic function is drawn on double logarithmic coordinate paper. 2. If the OD value of the sample is higher than the upper limit of the standard curve, it should be properly diluted and retested 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 E-selectin capture antibody, sample, standard product, biotin-labeled detection antibody, and HRP enzyme conjugate were added sequentially, incubated and washed in the middle, and colored with substrate TMB, which was converted into blue under the catalysis of peroxidase (HRP) and final yellow under the action of acid. There is a positive correlation between the depth of color and the E-selectin 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 | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human E-selectin ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | E-selectin, also known as CD62 antigen-like family member E (CD62E), endothelial cell-leukocyte adhesion molecule 1 (ELAM-1) or leukocyte-endothelial cell adhesion molecule 2 (LECAM2), is a selectin cell adhesion molecule expressed only on endothelial cells activated by cytokines. Like other selectin, it plays an important role in inflammation. E-selectin is encoded by the SELE gene. It has a cassette structure: an N-terminal C-type lectin domain, an EGF (epidermal growth factor)-like domain, 6 sushi domain (SCR repeat) units, a transmembrane domain (TM), and an intracytoplasmic tail (cyto). | |||||||||||||||||||||||||||||||||
| 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 | 0.15-10ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenates, cell lysates, cell culture supernatants and other biological fluids |
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4.5 ★★★★★
Based on 152 reviews
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Product Reviews
★★★★★ 3
A familiar story, just with…..less.
Format: Kindle
So, as other reviewers make clear, this is very similar to Pack Darling and The Beta. It’s much closer aligned with The Beta, in plot and maybe more like Pack Darling with characters.
That being said, I don’t hate this…..but it wasn’t great either. It’s both books mentioned but just….less. Less angst, less emotion, less feeling. The plot feels very half fleshed out, and the “bad guy” feels underwhelming. I didn’t really feel any real emotions from and of the male leads, except maybe Oliver. The others fell sorta flat for me. And Mika makes herself out to be this big bad ass straight outta training and then we never see it from here again with the one fitting room incident as the exception.
SPOILER:
The whole, “Oh, I’m actually probably an Omega, but I don’t wanna be but I do actually wanna be but no one can ever know my secret that I do nothing to hide “ thing fell so flat. She never commutes to believing she was secretly an omega, but also mentions her “secret” a lot. It just felt so manufactured.
I’m intrigued enough to read part 2 and see how the author closes everything out, but this is not one I’ll recommend or ever come back to.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on February 13, 2024
★★★★★ 5
Good start to a series
Format: Kindle
I delayed reading the series for reasons I don’t remember. But my TBR list is huge so I thought I’d take a shot of this and I was pleasantly surprised. I didn’t think the blurb about it was anything special. But it was a very good book. It took some interesting twists and turns. I am so glad the second book is already out. Because I would not have waited patiently. Very slow burn but good storyline. 🔥🔥/5
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Reviewed in the United States on January 3, 2025
★★★★★ 4
A good read
Format: Kindle
Multiple points of view. 3 Alpha men and an Omega male. She is a Beta in training for a new program placing betas in Alpha/Omega packs. Mila is only doing the program for the money to take care of her dad. She wasn't expecting to fall for a pack but when she sees this packs Omega she is done for. There is just something about him. His Alphas are good looking as well. Too bad she is hiding a secret and their government is acting shady. I liked it and can't wait to see where their story goes.
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Reviewed in the United States on November 14, 2023
★★★★★ 3
Slightly repetitive but I did love some things
Format: Kindle
I love this type of story. And omegaverse is one of my all time favorite genres. But there are a few things that pulled me out of my enjoyment while I was reading. It was repetitive at times as well as struggled with telling not showing. So we didn’t always feel like we were experiencing things with the main character. There were also some plot holes but they may still be answered in part 2.
Now this isn’t to be said I didn’t enjoy parts of the story. I loved the almost instant love between Mila and Oliver. And how he started changing around her.
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Reviewed in the United States on February 15, 2024
★★★★★ 5
delightful read
Format: Kindle
What a delightful read. The characters are awesome, the plot was so good, I loved it. I was intrigued and it kept me wanting more. Told in multiple pov, the book sucks you in and doesn’t let go. I cannot wait to read the next book.
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Reviewed in the United States on January 30, 2025