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Description
Rat XBP1 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: Adherent cells should be gently washed with pre-chilled PBS, then trypsinized and harvested by centrifugation at 1000×g for 5 minutes. Suspension cells can be harvested directly by centrifugation. Collected cells should be washed three times with pre-chilled PBS and resuspended 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. 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 utilizes 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 an X-Box Binding Protein 1 (XBP1) capture antibody. After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by HRP peroxidase and to yellow by acid. The intensity of the color is positively correlated with the amount of X-Box Binding Protein 1 (XBP1) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat X-Box Binding Protein 1 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | X-box binding protein 1, also known as XBP1 or TREB5, is a protein encoded by the XBP1 gene. The XBP1 gene is located on chromosome 22, while a closely related pseudogene has been identified and mapped to chromosome 5. This protein is a transcription factor that regulates the expression of genes important for the normal functioning of the immune system and cellular stress responses. It is a bZIP domain-containing transcription factor. It was first identified by its ability to bind to Xbox, a conserved transcriptional element in the human leukocyte antigen (HLA) DRα promoter. | |||||||||||||||||||||||||||||||||
| 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 | 0.156-10 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Tissue homogenates, cell lysates, and other biological fluids |
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4.2 ★★★★★
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Product Reviews
★★★★★ 5
This is a "Go-To" for thinking about Cloud Challenges.
Format: Paperback
Delivering and managing fully realized applications in the cloud is different. Different approaches to classic engineering problems than traditional On Premise development and different ways of thinking through the problems of "always available" solutions.
I've been in the software delivery business a long time, and with the cloud emerging, for good and ill: I understand the problems, but may be just a little set in my ways. I find this book helps me re-frame challenges in a way that aligns with the strengths of cloud computing. Solve the same problems faster, by thinking about them differently.
I'm finding "97 Things Every Cloud Engineer Should Know" great for re-centering my expectations about Cloud Native development and deployment of assets. I started reading it cover to cover over the Christmas Holiday but now i just pick it up and look for the group of essays about exactly the problem I'm wrestling with.
P.S. I'm heartened by the editors commitment to Black Lives Matter and Rule of Law. Mentioned only to balance the concerns from another review.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on January 24, 2021
★★★★★ 3
have some good contents but too general
Format: Paperback
The book covers some good points, but overall, it's too general.
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on June 28, 2024
★★★★★ 3
Why Politics in a Tech Book????
Format: Kindle
Well... I'm surprised to see the book blatently calls out its dedication to Black Lives Matter, which is in all caps so I assume it's referring to the political organization. It goes on to speak of 2020 being the year of an "awakening of injustices of systematic racism"... I thought I was buying a technical book??? Had I known this political bs was included I wouldn't have purchased it! However, I bought and I'm still reading it. If the politics goes away and the TECHNICAL content is good I'll update my review.
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Reviewed in the United States on December 13, 2020
★★★★★ 2
Not good use of time
Format: Paperback
It’s not clear who this book targets - neither experts nor novice will benefit. There are expert perspectives, only few of these are helpful, rest are too generic to be of any use. For instance the last entry is one an engineer who shares how she went from zero to expert in cloud engineering in six months but fails to mention a single resource or pathway for others to follow.
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Reviewed in the United States on April 2, 2022
★★★★★ 3
Uneven compendium of tips and insights, but still very useful
Format: Kindle, Format: Kindle
“In theory, theory and practice are the same. In practice, they are not" is why such bottom-up insights and lessons from the field are the fastest way to learn real life stuff. This series had a GREAT start with "Engineering Management" - I guess because it is way more subjective than Cloud Engineering and offered a variety of non-overlapping POVs. This one is a mixed bag, perhaps because "Cloud Engineering" was perceived amorphously by the authors. The scope was broad - from cloud-native (architecture), to cloud-ready (topology), to cloud-operations, to choosing tech (e.g., Lambda/serverless), to -ilities and economics -- it is like celebrating Halloween, Christmas and Labor Day together in a single long weekend.
I would give it 4/+ stars if at least 25% of such a book was "superb", giving 3 because about 10% of the book is. That still leaves 10 solid insights or learning that would otherwise take many failures to learn. And failures, especially in this emerging domain of complexity, is VERY expensive. Would love to see more books like this.
Let's summarize some key insights -
-- Real-time visibility across the entire DevOps lifecycle is key to winning in cloud.
-- Operations, especially operations at scale, is extremely hard. So, wherever possible, use Managed Services.
-- Distinguish between "availability" and "uptime" and measure each separately, and concretely.
-- In FaaS/Serverless, calling a function synchronously increases debugging complexity.
-- Good code is like good joke - it needs no explanation.
-- "Building your app or platform on top of the abstractions that a cloud provider gives you does not make the underlying layers stop existing. In many cases, it makes them even more important." That makes the failure modes LESS obvious than we were used to. Therefore having "extreme visibility" into your systems will help "separate the issues at the layer you're focused on from the fundamental system issues". i.e., just because what was under the hood is now even less visible, don't forget them. Many recent "cloud failures" have been in networking fault domains.
-- Cloud is not optimized for replacing static infrastructures.
-- Containers, service meshes and serverless jumpstart dev productivity but they also change the attack surface of apps and infra.
-- "Number of containers that are alive for 10 sec or less has doubled to 22%". 73% of all containers live for 30 minutes or less.
-- Adopt an "assume breach" stance for everything. Have a break-glass account.
-- Ensure you have a thorough understanding of where and how secrets are secured.
-- Grey failures (transient degradation of services) are often worse than complete crashes, since the latter have a short feedback loop.
-- Resilience engineering has existed as a sub-discipline within safety sciences. We just recently started applying its concepts in technology. Resilience can be thought of as a "socio-technical system" with Robustness ("system X has property Y that is robust in sense Z to perturbation W"); Reliability (consistent operations or service levels); Rebound (ability to deal with a chaotic situation using structures developed AND deployed BEFORE the chaos). In other words, robustness protects systems against a SPECIFIC type of failure mode. When a system is robust in many dimensions, it approaches good resilience to failure.
-- Resilience is something you "do", not something you "have". Resilience is a verb.
-- Moving from one class of nines to the next is 10 times more expensive.
-- Production System really means "system that someone else, anyone else, can hold you accountable for".
-- Most common theme across incidents is that something, somewhere was surprising.
-- Incidents are unplanned investments...your challenge is to maximize ROI.
-- We used to think of scale in two dimensions - horizontal (more) and vertical (bigger). In cloud, think of "scale out" (when demands increase) and "scale in" (when demand decreases).
-- Architecture diagram is also a map of failure modes.
-- Async communication is a friend of Cloud Reliability.
-- Test in production is a competitive advantage. The complexity of traffic patterns going through high-scale production systems is increasingly harder to reproduce in a controlled env.
-- Hundreds of open issues is fine, but if the repo has gone months (or, years!) without a release, THAT is a warning sign.
-- It is hard to write good tests for bad code.
-- Platforms come and go. But first principles and patterns will always exist, because they are the ones and zeros.
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Reviewed in the United States on November 6, 2023