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Description
Human TLR4 ELISA KitProduct Specification Usage Required experimental equipment: 1. Microplate reader (450nm) 2. High precision pipettes and pipette tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37C incubator 4. Distilled or deionized water Sample preparation and requirements: Tissue homogenization: Rinse the tissue with pre chilled PBS (0. 01M, pH 7. 4) to remove residual blood (lysed red blood cells in the homogenate will affect the measurement results). Weigh and
Product Specification
| Usage | Required experimental equipment: 1. Microplate reader (450nm) 2. High-precision pipettes and pipette tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37°C incubator 4. Distilled or deionized water Sample preparation and requirements: Tissue homogenization: Rinse the tissue with pre-chilled PBS (0.01M, pH 7.4) to remove residual blood (lysed red blood cells in the homogenate will affect the measurement results). Weigh and mince the tissue. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1g of tissue sample to 9mL of PBS. The specific volume can be adjusted according to experimental needs and recorded. It is recommended to add protease inhibitors to the PBS) in a glass homogenizer and grind thoroughly on ice. To further lyse tissue cells, the homogenate can be sonicated or repeatedly frozen and thawed. Finally, centrifuge the homogenate at 5000×g for 5-10 minutes, and collect the supernatant for analysis. Cell Lysis Buffer: Gently wash adherent cells with pre-chilled PBS, then trypsinize and collect the cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash the collected cells three times with pre-chilled PBS and resuspend in 150-200 μL of PBS per 1×10^6 cells (it is recommended to add protease inhibitors to the PBS; if the cell count is very low, reduce the PBS volume appropriately). Disrupt the cells by repeated freezing and thawing or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and collect the supernatant for analysis. Cell Culture Supernatant: Centrifuge at 1000×g for 20 minutes. Collect the supernatant for analysis, or store at -20°C or -80°C, but avoid repeated freezing and thawing. Other biological fluids: Centrifuge at 1000xg for 20 minutes, remove the supernatant, and test. Pre-test preparation: 1. Remove the test kit from the refrigerator 10 minutes in advance and equilibrate to room temperature. 2. Prepare the standard gradient working solution: Add 1 mL of universal diluent to the lyophilized standard, let it stand for 15 minutes to completely dissolve, then gently mix (concentration is 10 ng/mL). Then dilute to the following concentrations: 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, 0.15625 ng/mL, and 0 ng/mL. Serial dilution method: Take seven EP tubes and add 500uL of universal diluent to each. Pipette 500uL of the 10ng/mL standard working solution into the first EP tube and mix thoroughly to make a 5ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves as a blank well; there is no need to pipette liquid from the penultimate tube. See the figure below for details. 3. Preparation of biotinylated detection antibody working solution: Centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute 15 minutes before use. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration with universal diluent (e.g., 10uL concentrate + 990uL universal diluent). Prepare and use immediately. 4. Prepare the enzyme conjugate working solution: 15 minutes before use, centrifuge the 100× concentrated enzyme conjugate at 1000×g for 1 minute. Dilute the 100× concentrated HRP enzyme conjugate to a 1× working concentration with universal diluent (e.g., 10 μL of concentrate + 990 μL of universal diluent). Prepare immediately. 5. Prepare the 1× wash solution: Dispense 10 mL of 20× wash solution into 190 mL of distilled water (concentrated wash solution removed from the refrigerator may crystallize; this is normal. Allow to stand at room temperature until the crystals have completely dissolved before preparing). Procedure: 1. Remove the desired strips from the aluminum foil bag after equilibration at room temperature for 10 minutes. Seal the remaining strips in a ziplock bag and return to 4°C. 2. Sample addition: Add 100 μL of sample or standard of varying concentrations to the corresponding wells. Add 100 μL of universal diluent to the blank wells. Cover with a film and incubate at 37°C for 60 minutes. (Recommendation: Dilute the sample to be tested at least 1-fold with universal diluent before adding it to the ELISA plate. This will reduce the impact of matrix effects on the test results. The sample concentration should be multiplied by the corresponding dilution factor when calculating the final sample concentration. It is recommended to run replicates for all test samples and standards.) 3. Add Biotinylated Antibody: Remove the ELISA plate and discard the liquid without washing. Add 100 μL of Biotinylated Antibody Working Solution directly to each well. Cover with a film and incubate at 37°C for 60 minutes. 4. Wash: Discard the liquid and add 300 μL of 1x Wash Solution to each well. Let stand for 1 minute, shake off the wash solution, and pat dry on absorbent paper. Repeat this process three times (a plate washer can also be used). 5. Add Enzyme Conjugate Working Solution: Add 100 μL of Enzyme Conjugate Working Solution to each well. Cover with a film and incubate at 37°C for 30 minutes. 6. Washing: Discard the liquid and wash the plate five times as in step 4. 7. Adding substrate: Add 90 μL of substrate (TMB) to each well, cover with a sealing film, and incubate at 37°C in the dark for 15 minutes. 8. Adding stop solution: Remove the ELISA plate and add 50 μL of stop solution directly to each well. Immediately measure the OD value of each well at a wavelength of 450 nm. Calculating experimental results: 1. Calculate the average OD value of the standard and sample replicates and subtract the OD value of the blank well as a correction factor. Plot the standard curve of the four-parameter logistic function on double-logarithmic graph paper, with concentration as the horizontal axis and OD value as the vertical axis. 2. If the sample OD value is higher than the upper limit of the standard curve, dilute the sample appropriately and retest. Multiply the sample concentration by the corresponding dilution factor. |
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| Theory | This kit uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotin-labeled detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with a Toll-Like Receptor 4 (TLR4) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB converts to blue under the catalysis of HRP and to yellow under the action of acid. The intensity of the color is positively correlated with the amount of Toll-Like Receptor 4 (TLR4) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Toll Like Receptor 4 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Toll-like receptor 4 (TLR4) is a protein also designated CD284 (cluster of differentiation 284) with a molecular weight of approximately 95 kDa. TLR4 is a transmembrane protein and a member of the Toll-like receptor family, which belongs to the pattern recognition receptor (PRR) family. Its activation leads to the intracellular signaling pathway NF-κB and the production of inflammatory cytokines, responsible for activation of the innate immune system. TLR4 is expressed by myeloid cells (erythrocytes, granulocytes, and macrophages) rather than lymphocytes (T cells, B cells, and natural killer cells). Most myeloid cells also express high levels of CD14, which facilitates TLR4 activation by LPS. It is best known for recognizing lipopolysaccharide (LPS), a component present in many Gram-negative bacteria (such as Neisseria) and certain Gram-positive bacteria. Its ligands also include several viral proteins, polysaccharides, and various endogenous proteins, such as low-density lipoprotein, β-defensins, and heat shock proteins. | |||||||||||||||||||||||||||||||||
| General Notes | 1. Strictly adhere to the specified incubation time and temperature to ensure accurate results. All reagents must be at room temperature (20-25°C) before use. Refrigerate reagents immediately after use. 2. Improper plate washing may result in inaccurate results. Ensure that all liquid in the wells is aspirated thoroughly before adding substrate. Do not allow the wells to dry out during incubation. 3. Remove any residual liquid and fingerprints from the bottom of the plate, as this will affect the OD value. 4. The substrate developer solution should be colorless or very light in color. Do not use substrate solution that has turned blue. 5. Avoid cross-contamination of reagents and specimens to prevent erroneous results. 6. Avoid direct exposure to strong light during storage and incubation. 7. Do not expose any reagents to bleaching solvents or the strong fumes emitted by bleaching solvents. Any bleaching agent will destroy the biological activity of the reagents in the kit. 8. Do not use expired products, and do not mix components with different product numbers and batches. 9. Recombinant proteins from sources other than the kit may not be compatible with the antibodies in this kit and will not be recognized. 10. If there is a possibility of disease transmission, all samples should be managed properly and samples and testing devices should be handled according to prescribed procedures. |
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| Storage Temp. | If the unopened kit is stored at 4°C, the shelf life is 6 months. | |||||||||||||||||||||||||||||||||
| Test Range | 0.15-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, cell culture supernatants, and other biological fluids |
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4.7 ★★★★★
Based on 18 reviews
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Product Reviews
★★★★★ 5
A must read
Format: Kindle
A must read for everyone even remotely interested in self fulfilment and awakening. Can be read in an afternoon and echo for a life time.
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Reviewed in the United States on May 25, 2026
★★★★★ 5
A Beautiful Edition of a Timeless Journey
This deluxe edition transforms Coelho's beloved fable into a truly special keepsake. The larger format, quality paper, and thoughtful design details make it feel like the gift it's meant to be—whether you're giving it to someone at a crossroads in their life or treating yourself to an upgrade from a worn paperback copy.
The story itself remains as powerful as ever. Santiago's journey across the desert in search of treasure is deceptively simple on the surface, but Coelho's allegory works on multiple levels. Yes, it's about following your dreams, but it's also about learning to read the signs the universe offers, understanding that the journey itself transforms us, and recognizing that what we seek is often closer than we think.
I know some readers find the philosophy too simplistic or New Age-y, but I think that misses the point. Coelho isn't trying to be subtle—he's distilling universal truths into their essence. Sometimes we need straightforward wisdom delivered clearly, especially when we're overthinking our own paths.
This edition makes a perfect graduation gift, retirement present, or milestone marker for anyone embarking on something new. The story has earned its place as a modern classic, and this deluxe version does it justice.
"When you want something, all the universe conspires in helping you to achieve it." Still resonates.
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Reviewed in the United States on January 20, 2026
★★★★★ 5
Reading this book was an enlightening experience for me.
Format: Kindle
The Alchemist by Paulo Coelho provided hours of magical reading. It isn’t a plot-driven book, but rather a novel that provides a quiet sense of purpose and a deeper understanding of the universe. The period the story takes place isn’t clear, but it begins in Spain.
Santiago, a young man, perhaps in his teens, studied to be a priest, but realized what he really wanted to do was to be a shepherd, to move around, see new places. With the blessings of his family, he sets out with his flock of sheep and discovers not only the world, but himself. It soon becomes clear that what he searches for is an alchemist, a person who transforms things for the better.
Santiago meets people along the way who teach him many truths, some good, some evil, but each experience leads him closer to life’s true meaning.
This novel has an interesting history. “The Foreword” written by the author in 2014 speaks about the interesting path the novel has taken. The book was first published in 1989 in his native Brazil. Only one person bought a copy the first week of its release, then six months later another copy sold to the same person! His publisher cancelled the contract. Another publisher agreed to take the book and gradually had great success. An American read the book and wanted to translate the book from Spanish to English and find a publisher in the United States. It didn’t happen overnight, but the book became a phenomenon, has been translated into more than eighty different languages, and has sold thousands of copies.
Paulo Coelho never wavered in his faith in the book because it speaks of his own beliefs: A man sets out on a journey seeking a beautiful or magical place and then realizes the treasure lies within himself.
Reading The Alchemist was an enlightening experience for me. I highly recommend it for those who appreciate life’s purpose and who seek wisdom of the universe.
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Reviewed in the United States on October 7, 2025
★★★★★ 3
Good book, but I think the ending is a betrayal
Format: Kindle
As I write this I realize that I am talking about a modern classic, one that I must admit I did enjoy reading overall. But that said, I simply didn’t find this book to be as moving a book as I thought I would.
The novel tells a simple though interesting tale about a boy’s attempts to search for a treasure revealed to him in multiple dreams. Along the way he encounters many people and situations that enable him to learn and grow spiritually, aided by either his clever and inquisitive mind or the guidance of others who effectively serve as mentors.
For the most part, I did like THE ALCHEMIST. I enjoyed the simple style of writing and the stripped-down nature of the tale. We don’t get into the “five senses” kind of environmental description – Coelho’s tale is more of a parable at its heart, and in fact is a story OF the heart and of the mind. I also admired the central character as well: Santiago is not only aptly named, but he is a likable boy full of pleasant good will and a gentle disposition, ready to work and clever when it comes to the things one must do to succeed. He is also brave, and remains positive whether things are going well or they are not, choosing to do the best he can at whatever hand he is dealt by life.
Here, however, comes the problem. While I felt that the book worked for a good deal of the story, it weakened a bit toward the end. Part of it was the story’s mumbo-jumbo manner of religiosity; while I understood the author’s decision to tackle his subject in this simple, unadorned manner, it didn’t hold up at times. Certainly, the notion that there is a hidden one-ness at the heart of all matter is a notion that works well through a simplistic style, a la the clean, stripped-down beliefs at the heart of innocents or the way we conceptualize the purity of, say, the Native Americans culture when it came to nature. But when the boy began addressing the wind and other elements (a point I’ll get to further in a moment), it really broke down for me. It stretched credulity, reducing Nature to a kind of repertory theater. I will speak further on this scene in a second.
HERE I ONE ASPECT THAT WOULD BE CONSIDERED A SPOILER – SKIP THIS PARAGRAPH.
What I particularly disliked about THE ALCHEMIST is that I felt it broke its own rules. Again, I’m discussing the ending here, so beware. It bothered me that the ending should have been a point when the boy’s budding spirituality took its hold upon the lad. I ask: why would a person who is in essence learning so much about what really matters about life still keep seeking money? His interest in gold is not the same as the alchemist’s interest in it: the elder character is a man who can manipulate the elements in a near-magical manner, a process he leaves the boy to discover for himself (like when he fact almost comically abandons him to magically “make like the wind” for men who will otherwise kill him – comical because once the alchemist places the boy IN the situation, he simply goes off to play with his birds. And one more thing: while the twist at the end was clever, it disappointed me for two reasons: the first, for point I made above, but the second is trickier. The boy learns that the truth the material treasure HE sought is NOT there; it is, he learns, where the OTHER character says it is. Clever, sure… but is the author telling us the other character’s “information” is more true? Why is that? Was it that the other boy was favored? OR, which is probably more true, is it because whatever force it is that imparts such “wisdom” does so knowing that one boy WILL follow his heart and the other won’t. Think: if the other boy does follow his quest then Santiago’s info is going to be wrong. But IF things are meant to work out the way they actually do, then is Coelho indeed telling us that these actions were predetermined? This sends, I think, the opposite message the author wants to send.
THE ALCHEMIST is a book that people will enjoy for its simple values, and the tale of this young boy’s spiritual quest has satisfied many. While I too enjoyed it and liked the boy, I ultimately felt a bit disappointed in what I felt to be a contrived ending. Though I initially liked the twist at the end, the story could have ended on that exact moment and left me happier. As it is, I felt the final portion of the boy’s quest betrayed his AND the author’s intentions, which hurt the novel for me.
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Reviewed in the United States on October 4, 2016
★★★★★ 5
Actually get your skin clean rather than smudge with soap and then rinse
Size: Small
PROS
- I use a *lot* less water (including hot water) when showering now because I'm able to soap up with a little water and soap before I start showering.
- Definitely cleaner and healthier skin, feel like I need fewer showers.
- It's been hard to switch back to other types of soap -- this legit feels like the right way to do it.
CONS
- It took a shower or two to get used to the courseness of the pads.
- I have to scrub it against hard soap for a WHILE before it becomes smooth enough to apply enough soap that I can see it.
Overall a win and probably will buy again.
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Reviewed in the United States on March 21, 2026