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Description
Human TIMD4 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 T-cell immunoglobulin and mucin domain containing 4 (TIMD4). 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 T-cell immunoglobulin and mucin domain containing 4 (TIMD4) in the sample. The absorbance (OD) is measured at 450 nm using a microplate reader to calculate the sample concentration. | |||||||||||||||||||||||||||||||||
| Source | Human | |||||||||||||||||||||||||||||||||
| Synonym | Human T-cell immunoglobulin and mucin domain containing 4 ELISA Kit | |||||||||||||||||||||||||||||||||
| Detection Type | Double antibody sandwich method | |||||||||||||||||||||||||||||||||
| Composition |
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| Background | T-cell immunoglobulin mucin domain-containing protein 4 (TIMD4), also known as T-cell membrane protein 4 (TIM-4), is a protein encoded by the TIMD4 gene. It contains an IgV domain with an integrin-binding site and a unique metal-ion-dependent ligand-binding site for phosphatidylserine. It also contains a mucin domain with high levels of O-glycosylation. Unlike other TIM proteins, it lacks a tyrosine phosphorylation motif in its intracellular tail domain. Unlike other TIMs, which are primarily expressed on T cells, it is expressed on APCs, such as dendritic cells and macrophages. It serves as a ligand for TIM-1 and also as a receptor for phosphatidylserine. However, its phosphatidylserine binding does not mediate signaling, but rather functions more as a tethering receptor. It can also inhibit naive T cells through binding to non-TIM-1 receptors, but once T cells become active, it acts as an active regulator, helping to maintain their activity. TIM-4 expression on macrophages plays an important role in maintaining their homeostasis. It plays an important role in Th2 development. Therefore, it plays a role in the development of allergies. It is also considered a factor in tumor development. It also mediates autophagy at tumor sites, which leads to reduced antigen presentation and increased immune system tolerance to tumors. | |||||||||||||||||||||||||||||||||
| 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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4.2 ★★★★★
Based on 6 reviews
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Product Reviews
★★★★★ 4
Better than expected...
Style: For Him
I love going to a craft show or a place that sells a handcrafted soap. A good craftsperson can produce a high-quality soap that cleanses, moisturizes, and leaves a good scent. I gave these a shot after running out of those soaps.
They did beat my expectations. I thought these would be a factory produced and chemically. While you can tell these are churned out pretty regularly in some sort of factory, the overall quality is good.
Each scent is full and unique without being overpowering. They seem to be natural and not generic chemical smells. Really enjoy each of the unique scents.
The bars themselves aren't that big. They come in a piece of cardboard and are very uniform. There is some texture to the soap, as well as an added bit of something to give it some grit. I like it. This isn't lava soap but there is something (about the size of fresh ground pepped) mixed throughout each bar.
A drawback overall is how long they last. I would estimate less than a week for a daily user. I thought, given the denseness, they would last for a longer than they do. I'll probably use the entire box in under a month.
Of note is the lack of slimy or slipperiness to the soap. I like this. Some soaps that I've used in the past leave you slimy and you can barely hang on to the bar. This rinses very well and doesn't make you feel slimy.
Overall, these are a good replacement for the 'true' homemade soap. I am happy with them and would purchase them again.
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Reviewed in the United States on May 30, 2020
★★★★★ 5
Game changer!!!!
Style: For Him
Like a lot of folk who watch youtube I have been bombarded by Dr. Squatch soap ads over the last couple yrs. Couple that with my new mission of doing things as close to natural as I can, and my interest was peaked. I then did what most folks do when they want to learn more about said product, I went to the site and dropped a proverbial brick in my shorts when I looked at the price!!! I didn't want to give up on swapping the basement lab experiment that is my normal body wash for something more natural, but I also didn't want to sell one (or both) of my kids to do it. Enter crate 61. I didn't know this company from adam, they just had a "manly" variety box at a decent price with simple ingredients so why not. I'm here to tell you I'm never going back to "normal" body wash again.
I have the definition of combination skin. My father's skin was greasy, my moms skin is bone dry. Therefore for the first hour after my shower I have tumble weeds rolling across my forehead, and from then on, It feels like I headbutted an oil tanker. After a couple days of using this soap I felt 1000% more balanced. It's not a miracle in a bar, but I have never felt moisturized AND clean getting out of the shower in my whole life. Those effects last hours. Eventually I get greasy again but you can't fight nature regardless (this also does't claim too) but the point is I feel great. The main ingredients in all of the bars are a combination of olive oil, coconut oil, avocado and palm oil with other variations depending on the bar. Those good oils have been proven to have an anti inflammatory effect on the skin and while I wouldn't call it a cure for acne, my skin is much clearer. I chalk it up to simple ingredients vs chemicals in modern soaps. As far as the scents, It's a mixed bag and completely subjective. Beside I have never had a shower wash of any kind stick around for more than an hr anyway so It's a moot point. I for one dig some, not others, hence the variety pack but I'll know what to order going forward. The 6 bars do come in a very nice box and while it would be a fine gift as packaged, it tends to combine the smells until they are removed for a day or so. In regards to longevity, 1 bar has so far lasted 2 weeks with only half gone. I'm an average dude but I'm bald (i use it for hair soap too with no dandruff) and basically take military showers so millage may vary. I recommend using a loofa and turn the heat down on your shower just a little. If you use the bar directly on your skin in a hot shower it's probably going to disappear quick.
Honestly I'm ecstatic over this product and even recommended it to my sister who has the same dry vs greasy daily epic battle type skin as I do. The only downside so far is the eucamint bar I'm using now is not my fav and I want to move on but its holding up like a champ, go figure!
UPDATE: 4/12/20
I'm still not through the entire box of soap and it still feels great to shower with. I have 2 full bars left after all this time so longevity is a plus. I have noticed that using a loofa less is more. I actually get a better scent out of the soap if I don't go crazy with it, just a few quick rubs on the loofa and I'm golden plus I bought a soap saver and keep it on the opposite end of the shower head. In all fairness I shower about 2 to 3 times a week with Neutrogena acne soap because of the aforementioned greasy skin but this soap helps keep that soap from drying everything out too much.
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Reviewed in the United States on July 26, 2020
★★★★★ 5
Easy recommendation
Style: For Him
Actual reviewer, not paid to make this, nor was incentivized. Great value - these soaps last around 3 months each. Scents are non-offensive, soap doesn't leave my skin dry, and I feel clean afterward. They also lather up really well!
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Reviewed in the United States on May 4, 2026
★★★★★ 5
Great soap
Style: For Him
Luv this soap better than the Dr. Squash soap so far . It lathers good cleans ,no film , and luv the smell of it . I will most definitely buy again . It's my soap from now on for sure . Luv everything about it
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Reviewed in the United States on June 3, 2026
★★★★★ 3
IMMEDIATE REACTION, PLUS FOLLOW UP AFTER A WEEK!
Style: For Him
**Package arrived LITERALLY 5 minutes ago, here is my immediate initial reaction, and then I'll actually post this after USING the soap and getting a better feel for the product.**
Keeping in mind that smell is subjective, here are my knee jerk thoughts on the bars of soap:
- Firstly, I can smell the soap through the bag (and box). Whatever scent this is, it's POTENT.
- Opening the box, the bars of soap look a LITTLE small. Not egregiously so, but a little bit smaller than your average bar of soap; BUT I check them against a new bar of Dove soap that I have, and they are identical. The eyes are playing tricks on you with these.
- Here is the breakdown of the 6 scents
- Activated Charcoal. This is what I was HOPING would smell like the "Pine Tar" that other companies (like Dr. Squatch, which I've never used, btw) have, but it is the "soap-est" smelling soap, with a hint of tar. It smells a little bit like a can of new paint.
- Tango Mango. IMHO, the best smelling of the 6, with strong citrus smells. Very lemon-y, but pleasant.
- Eucamint. Ahhh, here's what was permiating out of the box. This smell is STRONG, with an AGGRESSIVE mint smell. Like, Ben Gay mint. And as strong to boot. It doesn't smell BAD, but it's hands down the strongest scent.
- Oatmeal Shea. Meh. Kinda smells like an old coat that would be hanging in your grandfathers closet. It's not a BAD smell, but it has this weird odor to it, like when you leave a can of peanuts in your cupboard for a long time, and then go back and smell it.
- Patchouli Lime. This one kind of smells like a mixture of Activated Charcoal, Eucamint and Oatmeal Shea. It smells like a room that you just painted a day or two ago, and it definitely has the Ben Gay after aroma lingering.
- Apline Spice. This smells like a box. Like find an empty cardboard box, open it, and take a whiff. Boom, Alpine Spice. My least favorite.
I will say, none of them STINK; as in there isn't a smell that I'm like, "ewww, I don't want to smell like AT ALL!" Which sounds weird, because I described some of them as "paint," "an old coat," and "a cardboard box," but I feel that those are just very passive smells, not aggressively bad ones. If there is a problem soap, I would assume it's going to be Eucamint, because it might just be too overpowering, although I've found that the more you smell them, they seem to mellow out. I imagine that after a shower with these, the smell will dissipate enough that it will be more mild. Still, with the exception of Tango Mango; a very good smelling soap mind you, I'd say they are all very "manly" scents, and I'm excited to try them out.
Ok, so I'm a little over a week in, and here's my final take. (I made it through Activated Charcoal and Tango Mango):
First, the scents ABSOLUTELY mellow out when used to actually shower with them. My wife, who admittedly isn't really up on me like that, hasn't even mentioned me smelling differently than the last 11 years. So, if she's even noticed, it's not on a level to make a fuss about. This is important because fresh from the box, these things have some powerful scents, but they are definitely tamed by washing with them.
The bars last ABOUT 7 showers. Obviously, your mileage will vary based on how long you're in the shower, how much you scrub, how you store them, etc, but I was able to get 7 GOOD showers out of these. I have them in these natural sisal (whatever that is) exfoliating bags that help lather and store the soap, and really did some good scrubbing in my showers. The bars lather decently, but I'm not sure how much the bag helps with that. If you are a once a day showerer, a bar a week is a very fair approximation of what you'll get.
I definitely felt.....SOMETHING I didn't before with my shower gel showers. I don't know if that's the moisturizing, or the exfoliation, but my skin felt a little different after showering.
A decent buy. I'm not thrilled, but I'm not upset either. I don't think it changed my life, or even the way I shower, but I'm not mad at the experiment. I may try the Dr. Squatch soaps to see how all these stack up.
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Reviewed in the United States on December 1, 2020