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Description
Human HIF-1a ELISA KitProduct Specification Usage Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High precision pipette and gun tips: 0. 5 10uL, 5 50uL, 20 200uL, 200 1000uL 3. 37 constant temperature box 4. Distilled water or deionized water Sample processing and requirements: 1. Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4 overnight, then centrifuge at 1000g for 20
Product Specification
| Usage |
Experimental equipment required for the experiment: 1. Microplate reader (450nm) 2. High-precision pipette and gun tips: 0.5-10uL, 5-50uL, 20-200uL, 200-1000uL 3. 37℃ constant temperature box 4. Distilled water or deionized water Sample processing and requirements: 1. Serum: Place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 4℃ overnight, then centrifuge at 1000×g for 20 minutes, and take the supernatant, or store the supernatant at -20℃ or -80℃, but avoid repeated freezing and thawing. 2. Plasma: Collect the specimen using EDTA or heparin as an anticoagulant. Centrifuge the specimen at 1000 × g for 15 minutes at 2-8°C within 30 minutes of collection. The supernatant can be assayed or stored at -20°C or -80°C, but avoid repeated freezing and thawing. 3. 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 the tissue and mince it. Add the minced tissue to the appropriate volume of PBS (generally a 1:9 weight-to-volume ratio, e.g., 1 g of tissue sample to 9 mL 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 the supernatant can be assayed. 4. Cell Lysis Buffer: Gently wash adherent cells with ice-cold PBS, then trypsinize and collect cells by centrifugation at 1000×g for 5 minutes. Suspension cells can be collected directly by centrifugation. Wash collected cells three times with ice-cold 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 freeze-thaw cycles or sonication. Centrifuge the extract at 1500×g for 10 minutes at 2-8°C, and remove the supernatant for analysis. 5. Cell Culture Supernatant: Centrifuge at 1000×g for 20 minutes. Remove the supernatant for analysis or store at -20°C or -80°C, avoiding repeated freeze-thaw cycles. 6. 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 capture antibody against Hypoxia Inducible Factor 1 Alpha (HIF-1a). 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 Hypoxia Inducible Factor 1 Alpha (HIF-1a) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Hypoxia Inducible Factor 1 Alpha ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Hypoxia-inducible factor-1α, also known as HIF-1-α, is a subunit of the heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1), encoded by the HIF1A gene. HIF1A, a protein containing a basic helix-loop-helix PAS domain, is considered a master transcriptional regulator of cellular and developmental responses to hypoxia. HIF1 is a heterodimeric basic helix-loop-helix structure composed of HIF1A, the α subunit (the protein), and the aryl hydrocarbon receptor nuclear translator (Arnt), the β subunit. HIF1A contains a basic helix-loop-helix domain near its C-terminus, followed by two distinct PAS (PER-ARNT-SIM) domains and a PAC (PAS-associated C-terminal) domain. The HIF1A polypeptide also contains a nuclear localization signal motif, two transcriptional activation domains (CTAD and NTAD), and an intermediate inhibitory domain (ID) that inhibits the transcriptional activity of CTAD and NTAD. | |||||||||||||||||||||||||||||||||
| 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 | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.6 ★★★★★
Based on 18 reviews
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Product Reviews
★★★★★ 5
Pom approved!
My pomeranian ONLY plays with small stuffies that squeak. She loves these and they are just small enough for her to hold and squeak. They are durable enough - but we will see when her big dog cousins come play with her, just how sturdy they are!
They are very cute, soft, and squeak well. Good value for the pack of them.
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Reviewed in the United States on December 2, 2025
★★★★★ 5
He loves his new toy and it’s funny!
Ever since I got this toy he can’t seem to stop holding one! Every time I look at him he always is has one in his mouth no matter the time of day. As you can see in my pics he is super proud and happy to go for a stress reliever after a hard day of being a dog. It’s honestly very entertaining and funny to watch and never fails to make me laugh. This item is super durable, and squeaks as he chews on it! It’s worth every penny if you ask me, perfect size and like I said super entertaining and looks super funny! It’s nice and soft so it won’t cause any damage on teeth would give this toy 10/10!
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Reviewed in the United States on June 3, 2026
★★★★★ 5
We are still laughing! Great toy
Fun, Interactive, and Great Quality Dog Toy
We purchased this dog toy and it has been an absolute hit in our home 🐶😄. As soon as we opened it, our dog was immediately interested. The main toy opens up and includes three individual “cigarettes” inside, which keeps the playtime going and makes it much more engaging.
We’ve had so much fun taking photos and videos because he carries it everywhere:
🛏️ to his dog bed
🛋️ to the living room while we watch TV
🛌 to the bedroom to stay close to us
The quality is excellent:
✔️ Durable, well-made materials
✔️ Strong stitching
✔️ Bright, attractive colors
✔️ Perfect size for carrying and playing
This toy is not only interactive but also very original compared to typical dog toys. You can tell it’s designed to stimulate curiosity and keep dogs entertained.
We are very happy with this purchase and would definitely buy from NestPark again. Highly recommended if you’re looking for a fun, unique, and durable toy your dog will truly enjoy 🐾💛.
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Reviewed in the United States on February 1, 2026
★★★★★ 5
Does your dog need a little something to take the edge off?
If your dog has a hard job of having to put up with a needy owner all day (me), this helps them step outside and take the edge off.
Seriously, my dog grabs this fake cig and suddenly she’s a 45-year-old trucker named Bernadette who’s seen too much, and only calls me on Christmas when she needs had a couple glasses and feels bad for the other 364 days of the year for being an absent mother.
These are cute and great quality. And honestly with the “pack” part and the three cigarettes, you technically get four toys. We’ve stuffed treats inside just for fun/enrichment and she enjoys snuffling them out (the pack opens from the top and can close). And plus with them being bigger, they fit our other dog who is larger and even our small dog gets a kick out of snatching one and running around the room like a madman.
This is a fun toy to buy and we would get it again. Plus it’ll be a cute Christmas gift to order for our dog friends who have a good sense of humor.
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Reviewed in the United States on November 14, 2025
★★★★★ 5
Funniest dog toy I own!
My dog loves these and my friends love them even more. They are funny, cute and the perfect size for my beagle. It did not last very long because he’s a chewer but great value for the money!
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Reviewed in the United States on June 5, 2026