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Description
Human HSPA5 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 20 ng/mL). Then dilute to the following concentrations: 20 ng/mL, 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.3125 ng/mL, and 0 ng/mL. Serial dilution method: Take 7 EP tubes and add 500 μL of universal diluent to each tube. Pipette 500 μL of the 20 ng/mL standard working solution into the first EP tube and mix thoroughly to make a 10 ng/mL standard working solution. Repeat this procedure for subsequent tubes. The last tube serves directly as a blank well; there is no need to aspirate the liquid from the penultimate tube. See the figure below for details. 3. Preparation of Biotinylated Antibody Working Solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute. Dilute the 100× concentrated biotinylated antibody to a 1× working concentration using universal diluent (e.g., 10µL concentrate + 990µL universal diluent). Prepare immediately before use. 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 Heat Shock 70kDa Protein 5 (HSPA5). After incubation and washing, the assay 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 Heat Shock 70kDa Protein 5 (HSPA5) in the sample. The absorbance (OD) is measured at 450nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Heat Shock 70kDa Protein 5 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Heat shock 70 kDa protein 5 (HSPA5), also known as binding immunoglobulin (BiP), is a protein encoded by the HSPA5 gene. BiP is a molecular chaperone of HSP70 localized within the lumen of the endoplasmic reticulum (ER). It binds to newly synthesized proteins during transport into the ER and maintains them in a state capable of subsequent folding and oligomerization. BiP is also an essential component of the translocation machinery and plays a role in the retrograde transport of abnormal proteins across the ER membrane destined for degradation by proteosomes. BiP is an abundant protein under all growth conditions, but its synthesis is significantly induced under conditions that lead to the accumulation of unfolded polypeptides in the ER. BiP comprises two functional domains: a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). The NBD binds and hydrolyzes ATP, while the SBD binds polypeptides. | |||||||||||||||||||||||||||||||||
| 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 | 0.31-20 ng/mL | |||||||||||||||||||||||||||||||||
| Applications | Serum, plasma, tissue homogenate, cell lysate, cell culture supernatant and other biological fluids |
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4.5 ★★★★★
Based on 16 reviews
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Product Reviews
★★★★★ 5
NIce Shoes
Size: 10, Color: Black
Very nice quality and comfort
WAS THIS REVIEW HELPFUL?YesReportShare
Reviewed in the United States on June 1, 2026
★★★★★ 5
Great shoe.
Size: 11.5, Color: Navy
Perfect fit. Stylish comfortable.
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Reviewed in the United States on May 2, 2026
★★★★★ 5
Comfortable shoes
Size: 10.5, Color: Black
Fit well Look great Get compliments all the time
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Reviewed in the United States on April 3, 2026
★★★★★ 5
Lightweight, comfortable, and elevated design!
Size: 7, Color: Grey
Ordered these for an upcoming trip to Italy where we will need to travel light so I wanted something I could push together in a suitcase where it might get squished - and I didn't want to do that with my expensive loafers/dress shoes.
These turned out to be the PERFECT choice - they are great shoes which fit true to size, feel light and pack extremely well, look dressy enough for dinner or the opera, but are still good to walk around in no matter what kind of streets we are walking on (Cobblestones and all, since places like Florence and Roma are ancient). They have a nice flexible upper and breathable knit which makes them light feeling on your feet too, they lace up nicely and stay tied, which is great, and they are just overall very comfortable shoes. I would have liked maybe a little more in the way of arch support since I have weak arches and that can be a problem for me when walking a lot...but I wore these as a trial beforehand just to make sure and no issues walking in them for well over a mile during a nice evening stroll post dinner. The inside does have a molded foam insert, so that's why I didn't take a star off for this - they might not have super high arch molds, but they are still very, VERY comfortable.
These truly do look like an oxford but feel like a sneaker, making them a great "go to" shoe for pretty much anything - you can wear these to work and then to go get chicken wings and watch sports at a bar and they fit in seamlessly in BOTH places, LOL. They look great with jeans or khakis, which is a plus. Note that I don't think these look dressy enough to wear to say, a wedding or something super formal, but that's only because they are knit and unless it's a casual wedding or at the beach, it's probably a little too unstated.
If packing these for a trip, know that you can put them in your suitcase for a long time and they pop right back up to looking like regular shoes without wrinkles or weird creases, and without ruining the shape. So they are great for a weekend trip where you really don't want to bring anything extra.
I got the gray pair and they are a nice in-between color that you can wear with anything. The black pair might seem a little more formal and dressier, but these are certainly no slouch, so if you aren't sure which color to get, the gray is a great choice.
And then there's the price. You cannot find shoes in this style which are hybrid work/play shoes for $32 these days, especially not new, even if you try your luck at the Nordstrom Rack or somewhere like that. You definitely won't find ones this nice and elevated looking either at a big box store - even Walmart charges for similar ones and they aren't nearly as nice. So these are a great value for the price and they should give you a lot of mileage for what is really not a lot of money. Time will tell how long they last but if they can survive the cobblestone streets of Italy for 3 weeks then they can probably make it through anything LOL. I will update this review accordingly after the trip and advise!
Highly recommend these if you need a dress shoe that's not stuffy so you can wear it with anything, or for a trip where you can't reasonably fit more than one pair of shoes, etc. They give you a great bang for your buck - you can't go wrong with them!!
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Reviewed in the United States on March 27, 2026
★★★★★ 4
Stylish price king
Size: 9.5, Color: Grey
Stylish and with the right insoles. Comfortable.
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Reviewed in the United States on April 9, 2026