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Description
Human TRA2b 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 uses a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Sample, standard, biotinylated detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with a capture antibody against Transformer-2 protein homolog beta (TRA2b). After incubation and washing, the sample is developed using the substrate TMB. TMB is converted to blue by peroxidase (HRP) catalysis and to yellow by acid. The intensity of the color is positively correlated with the amount of Transformer-2 protein homolog beta (TRA2b) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Transformer-2 protein homolog beta ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Transforming factor 2 beta (TRA2b), also known as splicing factor arginine/serine-rich 10 (SFRS10), is a protein encoded by the TRA2b gene. This gene encodes a nuclear protein that functions as a sequence-specific serine/arginine splicing factor, playing a role in mRNA processing, splicing patterns, and gene expression. It activates splicing of MAPT/Tau exon 10. It alters pre-mRNA splicing patterns by antagonizing the actions of splicing regulators such as RBMX. It binds to the AG-rich SE2 domain of SMN exon 7 RNA. It binds to pre-mRNA. Diseases associated with it include frontotemporal dementia and spinal muscular atrophy. It has been shown to interact with RBMX. | |||||||||||||||||||||||||||||||||
| 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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4.4 ★★★★★
Based on 25 reviews
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Product Reviews
★★★★★ 5
You will not be able to put it down
Format: Kindle
This is a why choose all-encompassing fantasy romance that captivates and holds one’s attention from beginning to end. It’s not surprising considering these two dynamic authors and their other bodies of work. Alyx is an assassin with reflexes like a cat and higher than average intelligence who has earned her reputation as the best around. Joha is the King of the land, but he is a figurehead squashed by the power-hungry Queen Mother, the woman who married his father but may have a surprise or two up his sleeve. Crux, is a king in his own right although he lives under the city (think tv show Beauty and the Beast with Linda Hamilton and Ron Perlman where there was an entire city below the city where people lived) and may or may not have a bit of a crush on a certain assassin. Orion is Joha’s right hand, a fierce bodyguard and friend. The way these authors have put this group together is nothing short of phenomenal. The storyline is filled with action, intrigue and spice. The way the alpha males obsess over Alyx is everything! The character development is so good that you feel part of the action. I highly recommend this book.
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Reviewed in the United States on September 14, 2024
★★★★★ 4
Quick plot twist
Format: Kindle
Ok so for starters I was obsessed with Crux from the start. An underworld boss? Sign me up. The obsession of love and protection he has for Alyx is a dream. Dark, protective, possessive, and obsessed ? Yes yes and yes. Love the character development from Our FMC. Alyx the Assassin that doesn’t back down and oozes confidence. Yes please again! I loved the slow cracks in her armor letting her feel love from all sides , everyone of her men gave her someone thing she needed. Joha kinda grew in to the king he needed to be but let’s be fareal he would have died tripping up the stairs if it wasn’t for Orion, Crux , and Alyx. Orions passion and dominance wooo. I love a good hard love making with a softy. Letting himself finally love Alyx but still be that rock of protection was very cute. WHAT I DIDNT like was the random plot twist with the atlas being alive?!? And then dying 5 minutes later. Spareeeeee me! That could have been a great story of the thrown being over taking , Alyx finding her way back. Her men bonding more and Joha finally being a badass and leading the riot/ rebellion to take his thrown back. I didn’t like the fast forward quick ending. Like oh yeah I was saved by sailor and poof here I am back from the dead and oh yeah brother take this slow stabbing in your chest. Nope . But overall this was a nice read . MMFM my fav.
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Reviewed in the United States on October 5, 2024
★★★★★ 5
Another win for this writing duo!
Format: Kindle
I loved this book.
I love everything about this book and these characters.
Alyx was such a strong female lead. She took no crap from anyone and was such a badass. I also loved that even though she was strong and independent, she allowed herself to be vulnerable when the tines were right, and i enjoyed seeing her growth throughout the story.
Joha had me obsessed from the very second he appeared on page. I adore his loving nature but appreciate that he did what he had to do in the long run. His innocent personality often had me swoonig for him. He is such a romantic and probably my favorite of the three.
Orion was a character i grew to love. At first, I wasn't crazy about him at all, but as the story progressed, so did my love for him. His sense of duty to protect his king and best friend made me respect him, and his character was well written.
And then there is Crux, another character that i was instantly intreagued in. I knew instantly i would love him, and i was correct. His possesivness, his willingness to do anything for Alyx, and his filthy mouth had me in a chokehold.
The plot of this book had me guessing what was going to happen all the way to the end. I didn't see some of the twists, which is hard as im very good at predicting outcomes. Im glad it was able to end the way it did. I was worried there for a little bit. But i adore the ending, and i would love to see more of these characters.
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Reviewed in the United States on September 16, 2024
★★★★★ 3
3.0
Format: Kindle
I enjoyed the story but found the rotation of chapters by characters broke the flow of the story. Choppy! Choppy!
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Reviewed in the United States on September 17, 2024
★★★★★ 4
A bit disappointed
Format: Kindle
I'm a huge fan of romantasy and I'm thrilled to see more RH romantasy books coming out. I also enjoy a good standalone and these are two of my favorite authors. Everything seemed like it should have added up to make a perfect book here...but I ended up feeling a bit disappointed.
The world is interesting and Alyx is the kind of FMC that I love. It's the lack of background on the male leads and, well, everything else that left me disappointed. Joha has a bit of a background story as it's central to the main plot..but Orion and Crux seem to have just popped into existence as full formed characters. I have no idea if they had families or friends before. Orion's entire personality is centered around protecting Joha and Crux's life is centered around Alyx. That's all I know about them.
Then there were some glaring inconsistencies. Atlas was Atticus earlier in the book. His backstory and how he planned everything is never explained. He just appears as the mastermind. There were no hints or lead ups that he might appear. It felt like the authors needed a villain to be the mastermind and he fit.
There were several other issues, but I won't drone on. Basically, it was a good book that missed the mark on being great. I think if it has been a trilogy or even a duet then the authors could have spent more time delving into the back stories of the characters and making everything feel believable. As it stands, it's good and I'll probably buy a hardcopy for my library but I don't think it'll be a book that I reread frequently.
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Reviewed in the United States on October 9, 2024