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Description
Rat PKCd 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, biotin-labeled detection antibody, and HRP conjugate are sequentially added to microwells pre-coated with a capture antibody against Protein Kinase C Delta type (PKCd). 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 Protein Kinase C Delta type (PKCd) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Rat | |||||||||||||||||||||||||||||||||
| Synonym | Rat Protein Kinase C Delta type ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Protein kinase C delta (PKCδ), also known as PRCKD or NPKC-delta, is an enzyme encoded by the PRKCD gene. The protein encoded by this gene is a member of the protein kinase C (PKC) family. PKCs are a family of serine- and threonine-specific protein kinases that are activated by the second messenger diacylglycerol. PKC family members phosphorylate a variety of protein targets and are known to participate in numerous cellular signaling pathways. PKC family members also serve as primary receptors for phorbol esters, a class of tumor promoters. | |||||||||||||||||||||||||||||||||
| 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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4.6 ★★★★★
Based on 26 reviews
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Product Reviews
★★★★★ 4
Well made shoes.
Size: 11.5, Color: Black
Four stars because of the weight. Too heavy for my complete comfort. Also I have a narrow foot (C width) and these are too wide for me. Heavy socks make them wearable but not as comfortable as I would have liked.
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Reviewed in the United States on December 1, 2025
★★★★★ 5
Good look
Size: 11, Color: Brown Crazy Horse
Great look.
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Reviewed in the United States on April 23, 2026
★★★★★ 1
Something's changed at Florsheim
Size: 11.5 Wide, Color: Black
I have a pair of size 11W Florsheim Palermo loafers that have lasted over 8 years and are still soft and exceptionally comfortable. Ordered a pair of 11W Florsheim Milano on Amazon but they are too small and have a cheap plastic sole. So, ordered 11.5W Florsheim Berkley and they were way too big. Returned them and ordered 11W Berkley instead and they were also too big ... and the Berkley also has a rubber sole instead of the original leather shown in the product photos. Guess they just don't make them like they used to.
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Reviewed in the United States on May 25, 2026
★★★★★ 3
Beware of the Brown Crazy Horse version.
NOTE: This review is COLOR SPECIFIC and concerns the Brown Crazy Horse version of this shoe.
I am a loyal Florsheim customer having purchased Florsheim Imperials in years past. They are a fine shoe. However, I have to admit complete frustration with the Florsheim Berkley Flex Penny Loafer in "Brown Crazy Horse." The photos in the listing show a shoe with gloss...a shine. The photos on the box show a shoe with shine. Don't believe the photos. "Brown Crazy Horse" is, at best a dull matte finished leather. If you want a classic penny loafer with the classic Florsheim look this is NOT the color to choose. When the shoes arrived and the shoes did not match the photo on the box, I ignorantly assumed it was merely a case of the wrong shoes in the box so I exchanged them for another pair, which was a big mistake. It is Florsheim's incorrect photo and box label that caused me the inconvenience of going through two pair and then having to return both. I would assume other colors are as pictured in their listings because I cannot imagine that every Florsheim Berkley Flex Penny Loafer is a dull matte finish. I'm sorry that I do not have photos of the dull finished shoes. You'll have to take my word for it. They are a matte finish at best.
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Reviewed in the United States on January 21, 2026
★★★★★ 5
Just right, great quality and value (in the price)
Size: 11.5 X-Wide, Color: Burgundy
Exactly what I'd expect from a Florsheim. I'm delighted.
Folks, let me share one mistake I made on my way to a good fitting pair of shoes. I ordered some in my size (I have a big foot, especially in width). I ordered what I thought I needed and it came in wide enough, but too short (the short was the surprise), but we all are told: "Manufacturers vary in their sizes not only from each other but from shoe model to shoe model." I want you to know I believe in that when it applies to clothes, especially children's and womens fashions in my life's experiences, but NOT with shoes, especially main-brand-deluxe. I ordered the next half-size up and now I have too large, how can that be? I found out when I pulled the paper stuffing out, in particular the LAST glob that extends from the middle to the toe, that it WASN'T the last glob, there was indeed a rather small glob still way down in the toe, almost pointless for this minor bit of stuffing being separate from the other, but it was. I had already innocently troubled Amazon with returns of shoes that would have fit, so I'll keep the half-size too large as a life lesson. I ordered this pair in the size I believed I was all along and viola, a good fit. What changed? When that last toe piece of paper wadding that's hard to see and even harder with a big hand to reach, the shoe fit perfectly. That's a lot of trouble because of a small piece of paper that was hard to see, but that's why I write this review for you so that you can make sure all the paper is really out when you already think it is. Amazon has been good enough not to hold me accountable and in return I'll not only repeat buy, I tell you of my experience.
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Reviewed in the United States on December 3, 2014
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