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Description
Human TRAF6 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 enzyme conjugate are sequentially added to microwells pre-coated with a TNF Receptor Associated Factor 6 (TRAF6) 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 TNF Receptor Associated Factor 6 (TRAF6) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human TNF Receptor Associated Factor 6 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | TNF receptor-associated factor 6 (TRAF6), also known as RING finger protein 85 (RNF85), is a protein encoded by the TRAF6 gene. The protein encoded by this gene is a member of the TNF receptor-associated factor (TRAF) protein family. TRAF proteins associate with and mediate signal transduction by members of the TNF receptor superfamily. This protein mediates signaling not only from members of the TNF receptor superfamily but also from members of the Toll/IL-1 family. | |||||||||||||||||||||||||||||||||
| 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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4.3 ★★★★★
Based on 30 reviews
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Product Reviews
★★★★★ 5
The finest hour of William Friedkin
William Friedkin was undoubtedly one of the 1970s greatest and most influential directors. He only made 3 pictures during the decade, but all are landmark films. The first was The French Connection (1971), the film that won him his best director Oscar & the film won best picture. His next film, The Exorcist (1973) is probably his most famous/infamous & and it’s the only movie that’s still a truly terrifying experience, something to endure as much as to watch almost lol. Sorcerer (1977) was his 3rd and final film of the decade, and in spite of or perhaps even because of how it was a largely forgotten film for so long (after seeing it on cable nearly 30 years ago, it was a movie I simply could not track down until the release of this Blu-Ray) I really think in so many ways it ranks right along aside his first two 70s classics, and perhaps even surpasses those films in terms of pure audacity. The rope bridge scene is as intense as a set piece gets and is worth the price of admission all on its own, but the film as a whole is a clinic in lean, economical filmmaking. And the score by Tangerine Dream kicks serious a**. It might be because of his 70s trifecta Sorcerer is the only one that is still almost sort of like a “new film” too me, as I’ve only watched it maybe 3-4 times and have seen the other 2 dozens of times, but for my money Sorcerer is master filmmaker William Friedkin at the absolute peak of his powers.
MD2020 gives “Sorcerer” 5 out of 5 ⭐️s and says it’s a flawless action film and Friedkins best work.
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Reviewed in the United States on January 25, 2024
★★★★★ 4
A stylish, intimate remake of a French Classic
William Friedkin (The French Connection) brings us exotic locations and gripping story in Sorcerer, a remake of Henri-Georges Clouzot's 1953 thriller Wages of Fear.
The first hour of this movie seems to be comprised of random events involving four individuals that have absolutely nothing to do with each other. The only commonality is that they are all men of dubious character. Probably the most recognizable actor in this film is Roy Schieder, who plays a low level New York gangster participate in a robbery that goes bad, and forces him to flee the country due to the fact he managed to step on some pretty well connected toes. The second man, a French businessman, escapes from the threat of prosecution due to some shady business dealings. The third man, a Middle Eastern terrorist flees his captors after being caught participating in a bombing, and the fourth man is a shady assassin type apparently on the run from repercussion from his last hit.
The key is all four men end up in a South American rat-infested stink hole of a town called Vera Cruz. After awhile, they find their sanctuary has become more of a prison and all begin a desperate search for a way out, but that involves money, a commodity that's in short supply, with the only work available in the area is provided by an oil company that runs a well and is building a pipeline for the bubbling crude. Dangerous work, and the pay is so low that it would take years to earn enough to leave. Salvation for the men comes in a catastrophic disaster, an explosion damaging the well and causing the oil to burn.
The only way to close the well proper for repairs is to blow it up, and the only explosives available are 200 miles away. Turns out the explosives, sticks of nitro, haven't been tended very well, and, in turn, are highly unstable. The company decides to hire four men to drive two trucks to carry the dangerous materials through 200 miles of dangerous jungle, including prehistoric rope and wood bridges, treacherous mountain passes, deadly bandits and swampy roads laden with fallen trees. The suicide mission draws the four men, as the money would allow them to escape the oppressive town. The most intense scenes for me involved the men trying to traverse a rickety, rotting wooden rope suspension bridge over a raging river during a heavy rainstorm. There were times when the truck on the bridge was at such an angle, I thought it would flip right off. And all the actors performed most all their own stunts, adding to the realism of the movie. Do they make it? See the movie and find out. I especially liked the very end. A very nice touch that reminded me of an ending of a certain Alfred Hitchcock movie. Which one? I won't say, as it would give it away.
The movie runs just under two hours, and I would highly recommend it to anyone. Friedkin and the actors do a wonderful job in developing tensions between the characters and the environment and the between the characters themselves. The pacing is a bit slow, but it's deliberate, cranking the suspense up, notch by grueling notch.
There are sparse special features, including pretty thorough productions notes and a trailer. If you enjoy thrilling adventure, you will most likely like this movie. I would have happily given this movie five stars but it is only available in the full screen format. Something I didn't understand is the title for the movie. I wasn't able to figure out what it had to do with the movie....
Cookieman108
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Reviewed in the United States on December 15, 2003
★★★★★ 5
Forgotten Classic
Sorcerer (1977) is, for the most part, a forgotten classic. First the forgotten: this film got kicked around by moviegoers when first released, probably because the title is misleading (nothing to do with fantasy), and they probably weren't in the mood for this film due to the sudden stratospheric success of another film that debuted at the same time (Star Wars). With Star Wars cementing the advent of the summer blockbuster, suddenly moviegoers wanted stuff more on the surface and eye candy for good measure. As a result, Sorcerer never got its footing, and was a major box office failure. If this film had debuted just a few years earlier, it would've done well, because it would've fit in with the other films of the time. The truth is that Sorcerer is an adaptation (not a remake) of The Wages of Fear from 1953. Now the classic: Sorcerer has since generated the respect it deserves, and is one of the best films of the 1970's. This film has it all: action, suspense, breathtaking and iconic scenes (the bridge scene being one), organic acting and a sort of 'raw' feel in terms of a lot of the settings; dwellings are dilapidated, and the machinery rusty. Even the actors are dirty, sweaty, and have visible wounds throughout the film. I particularly like the restraint on the dialogue, as Friedkin chooses to tell the story more in a visual sense. I also like the uniqueness of the characters being kept at a distance from the audience; this makes them sort of mysterious and seems to work in this film. This film was a very ambitious project, one that has been rewarded with the passage of time (thankfully). The care that went into many scenes and accompanying special effects are obvious, and first rate. Overall, Sorcerer is a must-see. This blu ray has excellent picture and sound quality, so it was worth the wait in that regard. One last thing: it's extremely rare for a blu ray disc to not include any special features; in the case of Sorcerer, it's unfortunately true. Perhaps this fact tells us just how much of a beating this film took over the years. I guess we should feel grateful that it has even made it onto blu ray.
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Reviewed in the United States on March 25, 2017
★★★★★ 5
Sorcerer (1977) (BD)
This is a different version, which can happen after the initial film with different editing and ending. I don't remember the political emphasis at all, and the ending of the other version was with Roy Scheider getting through the rough parts with his eyes glowing as he drove the truck alone, a real Sorcerer, after performing the miracle. This version continues after the completion to another ending altogether. I think both are great films, but each with a separate focus. This was all done without CGI which seems impossible. The scenes are still amazing and the camera work and editing top-notch.
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Reviewed in the United States on March 10, 2026
★★★★★ 3
Good but not Wages of Fear
The much maligned Sorcerer is in fact a good well crafted thriller that if seen without refrence to the Original Wages of Fear would have faired far better at the hands of the critics. That's not to say the film is perfect. The opening scenes that fill the viewer in on the charecters back stories, are pointless and actually work against the tension that is built up later in the film. Knowing exactly why the characters are desperate makes their reasons for taking on the deadly task seem a bit lame and looses the exestential angst that creates such a hopeless atmosphere.
Once in South America the oppresive nature of their stay in the village is also diminshed by knowing too much about them. Having said all that there is still an air of desperation and mounting frustration, it's just not as clausterphobic as it could have been. Once the journey begins the tension is turned on, the only problem is that by knowing the original so well there is not quite the frisson of that classic. This is an unfortunate by-product of re-making any well respected film. Friedkin however has a fair go and if this is the film you see first you will wonder why it was so slated on original release.
The film moves at a well constructed pace that is aided by a cracking score from Tangerine Dream. The cast perform well and Roy Scheider is reasonably well cast but does not deliver the subtly nuanced performance he is capable of, for this you need to look towards (obviously) Jaws and to a lesser extent the minor role he playes in Naked Lunch.
All in all a good film, though not great, and as the review starts by saying, not Wages Of Fear, but it deserves a better reputation than it so unfortunatly has and will be a good addition to anyones collection of interesting 70's films (the best decade for American Filmaking by a long long way).
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Reviewed in the United States on September 29, 2005