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Description
Human PKB 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 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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| Sensitivity | 0.14 ng/mL | |||||||||||||||||||||||||||||||||
| 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 protein kinase B (PKB) capture antibody. After incubation and washing, the assay 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 protein kinase B (PKB) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human Protein kinase B ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | Protein kinase B (PKB), also known as Akt, is a collective term for a group of three serine/threonine-specific protein kinases that play key roles in multiple cellular processes, such as glucose metabolism, apoptosis, cell proliferation, transcription, and cell migration. Three distinct genes encode protein kinase B isoforms, designated AKT1, AKT2, and AKT3, encoding RAC α, β, and γ serine/threonine protein kinases, respectively. The terms PKB and Akt may refer collectively to all three genes but are sometimes used to refer to PKB alpha and Akt1 individually. Akt isoforms are overexpressed in various human tumors and are genomically amplified in gastric adenocarcinoma (Akt1), ovarian cancer (Akt2), pancreatic cancer (Akt2), and breast cancer (Akt2). | |||||||||||||||||||||||||||||||||
| 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 | Tissue homogenates, cell lysates, and other biological fluids |
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4.6 ★★★★★
Based on 14 reviews
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Product Reviews
★★★★★ 4
Concise Book
Format: Paperback
I had read and felt her energy from her other book, Astral Travel for Beginners, and I loved that book! So, I bought this book. This book feels less personalized for me and more general but if you want a quick and concise book on this topic you might like that. I think a book of approximately 100 pages would be more reasonably priced at $7 versus $10 as well.
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Reviewed in the United States on November 19, 2024
★★★★★ 5
Great book!
Format: Paperback
I absolutely love this book! This is a very simple book but incredibly informatively written. Thoroughly enjoyed it!
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Reviewed in the United States on March 4, 2025
★★★★★ 5
Flavor-packed and accessible recipes
Format: Hardcover
This book delivers. It is structured sensibly, with categories for Snacks, Salad, Carbs, Surf & Turf, Chicken, Veg, Sandos (sandwiches), Breakfast, and Sweets. The ingredient lists are broken down by category, making it easy to identify the produce, dairy, protein, and pantry ingredients you need to assemble, and ingredient prep is thankfully built into the recipe directions, rather than the ingredient lists, so there's no time wasted as you cook.
The recipes are mouth watering and have relatively short but impactful ingredient lists, and are written in an accessible voice that helps the reader learn why certain choices were made. For example, the recipe for "Shells, peas & buttermilk" instructs: "Stir in a small handful of grated pecorino—this isn't a cheese sauce, so don't go too crazy here. We want to really taste the peas and mint—the cheese is there more as seasoning. The sauce should still be quite loose and brothy." These small notes help the home cook understand what the final outcome should look and taste like, beyond just the ingredient list and measurements. And the videos and audio cook-along notes are an entertaining addition that make you feel like you have Molly cooking alongside you in your kitchen—definitely an innovation over the many other cookbooks I own.
As Molly notes in the foreword, the recipes are all purposefully balanced with hits of fat, acid, and (for some) spice. Most of the recipes are not exactly healthy—many call for butter, cheese, heavy cream, and/or oil—but after cooking a few recipes already, I can say that these ingredients pay off in the final product.
There are a large volume of recipes in this book, which I prefer over more minimal books—you'll probably find something that sounds good to you. Recipes that stood out to me include Sizzled [store-bought] Dolmas with Yogurt and Brown-Buttered Pine Nuts; Sizzled Seedy Tomato Salad; Marinated Zucch & Mozz with Fried Sunflower Seeds; Drunken Cacio e Pepe; Grandma Pizza with Morty-D & Peperoncini Pesto [using store-bought pizza dough]; Broken Noodle Bolognese; Rigatoni with Creamed Leeks & Chive Bread Crumbs; Crispy Orecchiette with Spicy Sausage & Collard Ragu; Tangled Leek Pizza; Rarebit Mac 'n' Greens [why does a rarebit mac & cheese not exist already? brilliant]; Skirt Steak with Juicy Tomatoes & Salsa Macha; Crispy Salmon with Coconut Rice & Crackle Sauce; Hot Sauce-Braised Short Ribs with Winter Squash; Miso-Braised Chicken & Leeks; Chile-Braised Half Chicken with Caper Chimichurri; One Pot Chicken Mujadara; Curried Lentil & Sweet Potato Pot Pie; Spicy Green Fregola with Spicy Yogurt; Dilly Beans [canned white beans] & Burrata with Frizzled Shallots; Spicy Coconut-Smothered Green Beans; Sunken Drunken Apple Cake; Black Sesame Rice Pudding Brulee; Maple Ricotta Munchkins; Baklava Ruffle Pie... I could go on.
IMO this book is an improvement over Cook This Book, which featured good basic recipes but fewer innovative flavor combinations. I have fairly specific standards for recipes I actually want to cook—they should be doable within 30-60 minutes, include mostly ingredients I already have on hand, and promise better flavors than I could come up with myself—and nearly every page in my copy is dog-eared, which says a lot.
A minor note: The serif retro header font is difficult to read, the font size of the recipe text is small, and the page margins should be larger. But it's worth breaking out your readers.
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Reviewed in the United States on October 17, 2023
★★★★★ 5
More flavor, more fun
Format: Hardcover, Format: Hardcover
If you’re looking for a fun book with unique flavor combos, give More is More a go!
So far I’ve made the marinated zucchini, drunken cacio e pepe, shells peas and buttermilk, meatloaf, italian sandwich, tahini date shake, and the best cookies ever - the pistachio brown butter halva chocolate chunk cookies (a mouthful). Everything has been delicious and is on my re-make list.
Some highlights on my very long want-to-make list are her meatballs, broken noodle bolognese, chicken salad w coconut crunch & miso braised chicken.
Pros:
- Fun, creative recipes
- Recipes are written in a way that’s approachable and doable - helps build up my confidence and encourages me to try new things. Plus I know I can trust that the recipes are well thought and will work out
- Recipe pairing suggestions - this is a huge help for me personally and I’m already eating more veggies thanks to it
- Molly is very talented at creating a ton of flavor from a relatively short ingredient list
- The book has QR codes for audio and video follow alongs!
Cons:
- The header font can be hard to read, but the recipes themselves are readable
- The abbreviations can be a bit much at times, sure, but they’re just in the titles/descriptions, not the recipes themselves. Not a big detractor for me
I’ve seen some complaints that there are too many recipes from The Club. If the book had a bunch of recipes from a free site, I can see the argument for being mad about it. But there are only around a dozen recipes from The ($5/month) Club and if anything, I was happy they were printed in the book because I can now cancel my Club membership - was holding onto it because I didn’t want to lose some of my go-tos.
Molly has helped me gain confidence in the kitchen and become a more effective and efficient cook. Everything I’ve made from More is More has been fantastic so far and I’m super pumped to continue cooking through it!
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Reviewed in the United States on October 17, 2023
★★★★★ 5
From Boozy Pasta to Epic Meatloaf — A Hit!
Format: Hardcover, Format: Hardcover
This was the fifth cookbook we made in our cooking club, and I would say it has been my favorite (and the most fun) so far. Every recipe felt exciting and came with Molly’s playful twist on classics.
We cooked a full menu: the zucchini and mozzarella salad was fresh and flavorful, though zucchini can sometimes turn out a bit too soft or watery in texture. The Drunken Cacio e Pepe was creamy, delicious, and definitely on the boozy side! The meatloaf was an absolute hit, flavorful, juicy, and one of the favorites at the table. The chile-basted chicken was tasty, though a little too spicy for me. The clams with chili crisp and tofu were another flavorful surprise, while the cornbread was soft and comforting.
One standout is that Molly reinvents classics like meatloaf or cornbread in ways that feel new but still approachable. The spicy coconut-smothered green beans were so good I’ve already made them twice, once for my family, who were hesitant about the coconut, but ended up loving them.
Overall, this book delivers recipes that are exciting, delicious, and perfect for cooking with friends. For me, it’s a 20/10. Highly recommended.
*Español*
Este fue el quinto libro que hicimos en nuestro club de cocina, y diría que ha sido mi favorito (y el más divertido) hasta ahora. Cada receta resultó emocionante y con el giro creativo de Molly sobre los clásicos.
Preparamos un menú completo: la ensalada de zucchini y mozzarella estuvo fresca y sabrosa, aunque el zucchini a veces queda con una textura un poco demasiado suave o aguada. El Drunken Cacio e Pepe fue cremoso, delicioso y bastante boozy. El meatloaf fue un éxito rotundo, jugoso, lleno de sabor y de los favoritos en la mesa. El pollo con chile estuvo rico, aunque para mí un poco muy picante. Las almejas con chili crisp y tofu fueron otra sorpresa llena de sabor, y el pan de maíz quedó suave y reconfortante.
Lo mejor es cómo Molly reinventa clásicos como el meatloaf o el cornbread, haciéndolos nuevos pero aún accesibles.
Los green beans con coco picante fueron tan buenos que ya los he hecho dos veces, la primera para mi familia, que al principio dudaba del coco, pero al final les encantó.
En general, este libro ofrece recetas emocionantes, deliciosas y perfectas para cocinar en grupo. Para mí, es un 20/10. Muy recomendado.
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Reviewed in the United States on September 21, 2025