3/31/2024 0 Comments Incinerator toy story 3 sceneWhat then of Abraham, Moses and the other patriarchs who lived before Jesus' time but had died in the friendship of God? Must they burn in Hell because the taint of their Original Sin was not removed through the acceptance of Christ? There's simply no other way to get into Heaven. Limbo is a Catholic concept necessitated by the exclusive method of salvation in Christendom which is to be forgiven and brought back into a personal relationship with God by receiving Jesus' sacrifice. This is best read by people who have already seen Toy Story 3. The movie then show us - to my eyes, at least - the different possible fates of life after death some of which Andy's toys actually experienced for realsies while some were only described peripherally. As "being there for Andy" is pretty much their raison d'être, not being needed by Andy, by extension and for all practical purposes, represents the termination of their lives. The central plot of the film essentially talks about the toys' fate when their relationship with Andy, their child (now a teen and too old for toys), comes to an end. When I was watching Toy Story 3, I was positively bombarded with religious allegories and post-life metaphors - or maybe I'm just susceptible to to interpreting it that way, my being an amateur theology buff and everything. Both Toy Story films dealt with the themes of attachment and abandonment, but Toy Story 3 took it to its logical conclusion. It is probably the best film you can see in theatres for the time being until Inception premieres in July, and I am not ashamed to admit that the ending moved me to manly tears. It is also where Lotso is found and picked up by a garbage man who had once had another one like him when he was a kid.THIS IS NOT A REVIEW, but just to get it out of the way Toy Story 3 is Pixar's first threequel just as Toy Story 2 was Pixar's maiden voyage into sequel territory - and like Toy Story 2, Toy Story 3 proved to be better movie than its predecessor both in terms of storytelling and animation (I've seen it in 3D, but like Up, I feel the extra dimension adds little to the already 3-dimensional characters, so if you want to save a buck, seeing it in normal-D detracts nothing from the viewing experience). The claw carries the toys here and they, after rejoining with the aliens, hitch a ride home via Sid's garbage truck. Woody, Buzz, and the other toys end up here as a result of Lotso's betrayal and face certain, inevitable death until they are rescued by the aliens operating a giant claw. This is where garbage, cascading down an enormous bowl, is burned to ashes by an incinerator at the very center of the bowl. To avoid the shredders, the toys must grab metal objects that pull them to the magnetic ceiling.Īndy's toys as they almost go into the incinerator at the landfill. There is a magnetic ceiling that pulls metal objects from a bottom conveyor belt full of garbage. The shredders crush large objects into small chunks. Bulldozers plow garbage onto a conveyor belt that leads to the following locations below. Lotso, Woody, Buzz Lightyear, and the other toys get dumped here. This is where garbage trucks, property of Tri-County Sanitation, dump garbage from Tri-County Area.
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3/31/2024 0 Comments Emotional vocabulary wheelIn other words, as a child’s emotional vocabulary grows, that child’s emotional awareness and understanding will grow accordingly. It follows that the more terms available to describe emotions increase the potential to experience those emotions. Likewise, there could be a million different shades of happiness. but in essence, there a million different shades of blue. Colors are not limited to the names we give them: blue, red, yellow, green, etc. People’s emotions and feelings exist on a spectrum utterly independent of their labels or even identification. And beyond greater discernment, there is perhaps an even more significant benefit of the child developing an emotional vocabulary. However, the ability to understand abstract concepts catches up fairly quickly it begins to develop close to the child’s second birthday, with a rapid increase in the third year of life.Īs a child’s vocabulary expands, it becomes progressively more natural for the child to discern different emotions that are being felt. It is typical for children who are developing their vocabulary to first learn words that label tangible objects before learning those words that describe abstract concepts. This is because words that describe emotional states improve a child’s ability to comprehend abstract concepts. In language acquisition, emotion words in particular are pivotal in developing emotional intelligence. It has been found that emotions play a critical role in acquiring language. The Benefits of a Strong Emotional Vocabulary Developing Emotional Awareness While most people might say that they feel “bad,” those who are emotionally intelligent can identify whether they feel “irritable,” “frustrated,” “downtrodden,” or “anxious.” There is a direct correlation between the specificity of word choice and the degree of insight into exactly how you are feeling, the cause, and how you should react. This understanding allows those with high EQs to develop an extensive vocabulary of feelings which helps them. People with high EQs (like IQs but for Emotional Intelligence) can master their emotions because they understand them. Leaving feelings unlabeled allows them to go misunderstood, leading to irrational choices and counterproductive actions. Online school psychologists can often be of help.īut refraining from putting words to emotions can be costly. And finding the proper descriptor to express how you feel can be very challenging. At the same time, feelings and emotions themselves are complicated. Bradberry and others have found that having a useful emotional vocabulary increases your emotional intelligence. Understanding words and how to use them plays a vital role in emotional intelligence. Travis Bradberry, co-author of Emotional Intelligence 2.0 and president of TalentSmart, in reviewing the data of over one million people who have tested with TalentSmart, found that knowing how to express yourself is an essential key to emotional intelligence. However, its importance doesn’t stop there, as there is another equally critical component as well of which online school psychologists are well aware. Emotional intelligence is crucial in guiding us in our day-to-day social interactions with others, behaving appropriately, and understanding people’s feelings. 3/31/2024 0 Comments Winrar vs winzip winrarOof!ħZ added insult to injury: File Explorer took nearly nine minutes to extract, while WinRAR and NanaZIP finished in about one minute. RAR was next, and it immediately tripped File Explorer, causing it to complete the process in almost six minutes, more than three times slower than WinRAR and two times more than NanaZIP. The experiment revealed that File Explorer had no problems extracting TAR archives and could keep up with WinRAR and NanaZIP: all three finished in about 80 seconds, an unsurprising result considering the nature of TAR format. Keep in mind that my goal was to see the performance difference between apps, not to find another reason to upgrade my computers, which I did anyway since the Zen+ architecture is starting to show its age, and I need a better CPU for the upcoming Forza Motorsport release. Then, I repeated the process on my laptop with the Intel Core i3-1125G4, 16GB DDR4-3200, and a 500GB NVMe SSD. Now, it is time to unpack the archives and measure the time it takes for Windows 11's File Explorer, WinRAR, and NanaZIP to complete the job.Įach app went through three tests to get the average result on my desktop PC with the Ry(I will upgrade to 5600 in a few days), 32GB DDR4-3200, NVIDIA RTX 4060, and Samsung 980 SSD 500GB. I downloaded Microsoft's massive 24GB free Windows 11 virtual machine and packed its files into three previously unsupported formats: RAR, 7Z (50% compression rate), and TAR. To answer the question, I decided to test the updated File Explorer versus WinRAR, arguably the most popular app of its kind ( which is honored by the change), and NanaZIP, a great fork of 7Zip for Windows 11. Still, the change raises a question: Can the updated File Explorer replace WinRAR, 7Zip, NanaZIP, and other dedicated apps for those frequently working with archives? Even though there is no shortage of free and premium file compression apps, native support is always better for the average consumer-you do not need to look for a program to extract a single randomly downloaded archive. Therefore, users had to opt for third-party apps to work with RAR, 7Z, etc. Although there are concerns about performance and bugs, in this article, I want to focus on the positive side and look closely at one of the best new features the redesigned File Explorer will introduce: native RAR, TAR, 7Z, and other file compression format support.īefore Windows 11 version 23H2, ZIP was the only natively supported archive format. Version 23H2 is expected to introduce many quality-of-life improvements and new capabilities, including a File Explorer redesign. Our employees do not have comp.Microsoft plans to release a big feature update for Windows 11 later this year. I am looking for an alternative that can either use a landline or a desktop app. We have been using Authy's desktop app for a few years, but it is going EOL next month. Authenticator app that doesn't require a cell phone SecurityĪ few of our clients require MFA to access their sites.Just a reminder, if you are reading the Spark!, Spice it Hope you are all enjoying some spring like weather as well. Sure is nice around here, feels like spring. Spark! Pro series – 23rd February 2024 Spiceworks Originals.It was created hastily just so I could make some posts, so the quality isn't the best. Suzanne (Spiceworks) asked me to copy the Spark! post I made on Load-Test day in the Community Playground so it would be preserved. Spark! Load-Test Series 21-February 2024 Spiceworks Originals.Would you rather be a magician or a mime if you were guaranteed success either way?2. Here are some questions to think about rolling into those favorite days of the week.1. Heck of a week □, about to be a heck of a weekend□. Stupid Friday ?'s | 2-23-24 Water Cooler. 3/31/2024 0 Comments Sight words 1st grade worksheetIdentify and read the sight word in the context of a sentenceĪre you ready to make your custom sight word worksheets? Step 1.Fill in the missing letters in the sight word.These worksheets follow a consistent format to foster student independence. So now that you know they are magically made…here is what the finished product looks like! Plus, you can create an unlimited number of sight word worksheets! The Worksheets Include students’ names and/or relevant content on every page.Īs you type your information into the table, the worksheets are INSTANTLY generated!.You can use these worksheets for ANY sight words, as long as they are between 2-9 letters.These worksheets are editable and they auto-fill. Just in case you didn’t watch the short video, here’s what makes these worksheets so magical! Take a sneak peak into the magic via the video, or keep scrolling to read more about it! Dolch sight words, Fry sight words, custom district sight words…Forget comparing sight word lists to see if you can use a particular resource! Forget about buying different sight word packs because Johnny is three grade levels ahead and Sally needs last year’s list. Ok, not really! □ But you don’t need to worry about which list you use. The magical part about these sight word worksheets is that they are efficient for you, too! Editable Sight Word Worksheets These sight word activities provide kindergarten, first grade, and second grade students with effective and engaging sight word practice. Editable sight word worksheets are just what you to need to efficiently create sight word printables for your students! They are so easy, you can actually sit back and relax this weekend! □□ The application helps to install and familiarize yourself with the mod for the game Minecraft, this is not a game, but an add-on with instructions! If you feel there are trademark violations which don't fall under the "fair use" rules, please contact us by email. Name, brand and assets are the property of the owner Mojang AB. Treasure and loot with unique traits and functions. Varied boss battles with elaborate mechanics. Some of the major features include: Fully Fledged dungeons. Many of them are exclusive and you can install them!ĭISCLAIMER: This is an unofficial app. Twilight Forest is a dimension exploration mod focused on adventure that will take you on a journey to meet strange creatures, exploring dungeons, and much more. Our application contains bonuses especially for you. Sie fügt viele neue Inhalte wie Gegenstände, Biome sowie Kreaturen hinzu. A realm basked in mystery and eerie twilight, you will overpower terrifying creatures and secure the adventure of a lifetime in the Twilight Forest. Always the latest version of the Twilight Forest addon and support for errors in installation and downloading Changes since last beta: - Fixed a server crash with the ice bomb entity. Changes since last beta: - Fixed a load order issue with Immersive Engineering, causing the game to fail to start. The mod is absolutely free and the Twilight Forest addon is installed instantly This Twilight Forest Mod supports the latest versions of minecraft and multiplayer! You can play Twilight Forest with your friends and immerse yourself in the wonderful atmosphere of the Minecraft PE game Zekocraft Texture pack is compatible with this mod.Twilight Forest Mod for Minecraft PE will transform your minecraft and add new emotions to you, unlike the old and Twilight Forest minecraft.ĭownload and install our addon for an unforgettable experience of travel and battles with the Twilight Forest mod.– Drag and drop the Mod’s files in Mods folder in Minecraft.jar Bring you house to life - added the ability to harvest torchberries from torchberry plants and ripe trollber, finally. – Open Minecraft.jar using any archive software (for e.g. To change it, add or remove fireflies from the jar. – Now go to Start > Run > Type “%appdata%/.minecraft/bin”, hit enter. A realm basked in mystery and eerie twilight, you will overpower terrifying creatures and secure the adventure of a lifetime in the Twilight Forest. The TWILIGHT FOREST Minecraft Mod Showcase - The FINAL BOSS, All Mobs, All Bosses & Every Biome EXPLAINED Full Guide & SpotlightIn this Twilight Forest Min. A magic map works much in the same way as a map in the Overworld does but with key differences. – Download the Twilight forest files from download link above. Magic Maps are an essential tool to any serious explorer in the Twilight Forest, as it links intrinsically to the dimension to show valuable information of the surrounding lands around the bearer. The Twilight Forest is a very popular and impressive mod which adds an entirely new dimension: the titular Twilight Forest. – Back up your previous Minecraft file(s). Download: The Twilight Forest Compatible with: Forge Requires: Minecraft 1.20.2. Throw a diamond in the pool (Hotkey: ‘Q’) and stay back.ĭescription: This mod adds a new teleportable world in Minecraft.Ĭompatibility: Windows, Mac, Linux Download Twilight Forest Mod:įor 1.7.10 – (Direct) TwilightForest-1.7.10-2.3.7.jarįor 1.6.3 – twilightforest-1-20-4.zip (coming soon) In order to do that, make a 2×2 Shallow pool (4 squares) of water in a grassy area, and cover it completely with natural habitat (Flowers, mushrooms or tall grass can do the job). With this mod installed you will feel bumped into a mystical world with completely. As the title suggests, the mod adds a unique dimension to your game, covered in the forest all over the place with tall trees reaching the sky. Fixed the goals we add to vanilla mobs not respecting line of sight. Designed, coded and textured by Electroblob. Twilight Forest is known as one of the most popular dimension mods for Minecraft for long years. Fixed structure mob spawns causing a lot of TPS lag in big modpacks that add lots of structures. An addon for Electroblobs Wizardry, a modification for Minecraft by Mojang AB. To enter the majestic forest of this beautiful mod, you need to create a portal. Curse Maven does not yet support mods that have disabled 3rd party sharing. Electroblobs Wizardry: Twilight Forest Spell Pack. Here's a look at SportsLine's top three 2022 Fantasy football WR picks:ġ. Ekeler had over 1,500 total yards last season to go with 20 touchdowns, and will be asked to do it again for the Chargers. After the injury struggles Christian McCaffrey has had the past few years, Ekeler could firmly establish himself as the top all-purpose back in the league this year. Austin Ekeler, Los Angeles Chargers: The Chargers didn't go out of their way to add many skill players to the offense in the offseason, which means Ekeler should once again be a focal point. Henry should be the stabilizing presence in Tennessee's offense and return with elite volume this year.ģ. Derrick Henry, Tennessee Titans: One of the most dominant running backs over the last few seasons should be fully recovered from the foot injury that kept him off the field for the back half of last year. New Colts QB Matt Ryan targeted running backs 26.3 percent of the time last season with the Falcons, and Taylor caught 40 passes in 2021.Ģ. Indianapolis head coach Frank Reich will prefer to not have to give him another 332 carries this season, but he should be able to make up ground in the passing game. Jonathan Taylor, Indianapolis Colts: Taylor showed that he could singlehandedly power the Colts' offense last season. Here's a look at SportsLine's top three 2022 Fantasy football RB picks:ġ. However, he's also thrown for 8,766 yards and 78 touchdowns while completing 65 percent of his passes over the last three years. Lamar Jackson, Baltimore Ravens: The former Heisman Trophy winner at Louisville has provided rushing productivity that would make him a legitimate flex candidate even if he never threw a pass, with 3,673 yards and 21 scores over four seasons. He's now averaged 4,677 yards and 38 touchdowns per season over the last four years and has the potential to throw for well over 5,000 yards and 50 scores in any given year.ģ. Patrick Mahomes, Kansas City Chiefs: Mahomes faced questions about his decision-making during a turnover-prone first half of the 2021 season, but threw just three interceptions over his last nine games and finished with strong numbers again. Over the last two years, he's completed 66.1 percent of his passes for 8,951 yards and 73 touchdowns while adding 1,184 rushing yards and 15 rushing/receiving touchdowns. However, his improvement as a passer from Year Two to Year Three has cemented him as the top Fantasy option. Josh Allen, Buffalo Bills: With better-than-expected speed for his size and a punishing running style, Allen's running ability and goal-line usage provides a solid baseline for Fantasy productivity. Here's a look at SportsLine's top three 2022 Fantasy football QB picks:ġ. Now, SportsLine has simulated the entire NFL season 10,000 times and released its latest Fantasy football cheat sheets 2022 and draft advice, along with its top Fantasy QB, WR, RB picks. The projections update multiple times daily, so you're always getting the best Fantasy football advice and the latest Fantasy football tiers. The model is powered by the same people who generated projections for all three major Fantasy sites, and it beat human experts last season when there was a big difference in ranking. Anybody who banked on players like those likely made a run at their league title. Brown in 2020, Henry in 2019, McCaffrey and Alvin Kamara in 2018, and Davante Adams in 2017. It's been high on Fantasy football sleepers like A.J. The result: Taylor led the NFL with 1,811 rushing yards and 18 rushing touchdowns to finish as the overall RB1 in 2021.That's just one example of a league-winning pick that SportsLine's model has been high on. Taylor was being drafted behind running backs like Aaron Jones, Ezekiel Elliott and Ekeler but the SportsLine projection model predicted that he'd outproduce all of those backs and be a fixture in your Fantasy football lineups. Last year, the model accurately predicted that Colts running back Taylor was being drastically undervalued. If you're looking for 2022 Fantasy football advice, be sure to check out the 2022 Fantasy football cheat sheets from the proven computer model at SportsLine. With the growth in popularity of points per reception and half-PPR leagues, running backs who can factor into their teams' passing games have seen a bump in value, sending them up the Fantasy football rankings 2022. With just days before the start of the NFL season, you need to figure out where you will prioritize your 2022 Fantasy football QB, RB and WR picks. However, several of the top players in the 2022 Fantasy football RB rankings also carry question marks heading into this year. Finding bellcow running backs to anchor your 2022 Fantasy football rankings is a must, no matter how much talent you have at other positions. 1st studio siberian mouse скачать, 1st studio siberian mouse hd 93, 1st-studio siberian_mouseġSt Studio Siberian Mouse HD-93 ->->-> ġst Studio Siberian Mouse Hd interdiction kazaali ,1st-Studio. 1st Studio Siberian Mouse Masha Veronika Babko - Fotoavi MB.1St. siberian mouses 1st studio siberian mouse full 11 Jan avi. Search torrent: 1St Studio Siberian mouse HD-93 video HD Masha. 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3/31/2024 0 Comments Black bear diner nutrition menuHe was honored by that organization with the Patriot Award for a lifelong commitment to our military veterans. Lance also serves on the Founder’s Circle Advisory Board for Project Healing Waters, which is dedicated to physically and emotionally rehabilitating wounded or disabled military service personnel. He has also been named to the Society of Fellows of the Culinary Institute of America.Ĭommitted to giving back, Lance served as a founding board member for Camp Corral, a summer camp for children of wounded or fallen military service members, and chaired the board in 2019. In addition, Lance was the recipient of the 2019 Chairman’s Award for the National Restaurant Association Educational Foundation and was the National Restaurant Association’s 2020 Chairman’s Award recipient for outstanding leadership and contributions to the industry. In 2019, Lance was named the Restaurateur of the Year by NCRLA and was a past recipient of the Griff and June Glover Award for Distinguished Service. Lance was the 2022 recipient of the highest honor in the restaurant industry, the IFMA Gold and Silver Plate Awards. He has chaired the Restaurant Industry Executive Council and served as a WFF CEO Advisory Council member. Lance also serves on the Executive Committee of the North Carolina Restaurant and Lodging Association and is a former Chair (2016). Lance’s industry leadership includes his current role as Chair of the Board of Directors for the National Restaurant Association, as well as serving as a Trustee for the National Restaurant Association Educational Foundation. This year marks Lance’s 36th year with Golden Corral. He was named President and Chief Executive Officer in January 2015, leading America’s largest buffet and grill concept and becoming only the third person to hold this position in the brand’s 49-year history. After deciding to pursue a multiunit career with Golden Corral, he quickly rose through the system, holding leadership roles of increasing responsibility. Lance joined Golden Corral in 1986 as Partner Manager. To learn more about Black Bear Diner, check out their website.Lance Trenary began his restaurant career in his father’s restaurant at a young age, where he learned the value of guests and the people who serve them. Foodįit & Focused Chicken Apple Sausage ScrambleĬubs’ Brandi’s Favorite Macaroni & Cheese Here’s a look at the latest Black Bear Diner menu prices. The restaurant has also received numerous awards recently and over the past years such as The Top Food Franchise of 2019, Fastest Growing Chains 2018, and many more. Once they sign up for The Black Bear Diner email list, they will receive a free birthday coupon.īlack Bear Diner has also done quite a bit in the community by donating a certain percentage of the company’s sales income to a children’s hospital called Valley’s Children. Their menu pattern looks quite similar to an obsolete newspaper which was named “The Black Bear Gazette.” Black Bear Diner offers to its customers a free meal for their birthday. Just like the name, each Black Bear Diner has a carved 12-foot tall black bear at each restaurant. Their opening hours are mainly from 6 a.m. They opened the very first restaurant in Mount Shasta, California, and they serve a variety of burgers, salads, and shakes, but the restaurant focuses more on a breakfast style menu and also sells alcohol at certain locations.īlack Bear Diner has 132 restaurants in the United States, and they plan on opening other restaurants very soon. Black Bear Diner is a restaurant that was founded in 1995 by Bruce Dean, Bob, and Laurie Manley. If you did want an itinerary of what to see in Middle-earth, the server has the perfect tour for you. That's around the same size as Dallas, Texas, so it's understandable that the team might not want to tackle the wider realms of Tolkien's universe quite yet. The Minecraft Middle-earth map is currently 29,000 by 30,000 blocks, which makes an area of around 870 square kilometres. That kind of stuff we haven't touched yet." "The Hobbit actually takes place in a different location, so we haven't built areas like Lake-town or the location where the Battle of the Five Armies happens. Visitors would ask, do you guys have Erebor built? And then we'd have to tell them no or, do you have Mordor built? And we would have to say, not yet." So of course, we had to disappoint a lot of people. Browse and download Minecraft Hobbit Maps by the Planet Minecraft community. "But we hadn't really built anything that appeared in The Hobbit except for a couple of places like The Shire or Rivendell. Minecraft Middle-earth was live when the Hobbit trilogy released between 2012-2014, and I ask Vermeersch if the server got excited to see new parts of Tolkein's world visualised. "When the first Hobbit got released, we saw a huge increase in visits for people," he says. The Middle-earth builders also need to apply their own creativity to vast parts of the map that Tolkien only alludes to. The references that the team use are the descriptions from the books and how landscapes are shown in the films, but Vermeersch also says that the group use their own fantasy concepts for parts of the world that don't work in Minecraft. Then we look around at the builds and find out what works and what doesn't work." We give them guidelines and present them with pictures of like, for example, Roman architecture, as kind of a mood board. We set it in a blank world and ask, for example, for houses in Minas Tirith and people begin building. "We then organize built contests, basically, where everyone can be involved. "The biggest, most iconic builds are usually handled exclusively by senior members or our best builders," Vermeersch says. Read about how it took 10 hours of walking to retrace the Fellowship's journey in Minecraft Middle-earth. Authors Developersĭylanhugh and Angmarzku for their ideas & arts for Gundabad and more.(Image credit: Minecraft Middle-earth / Mojang)Įver wondered if it was possible to walk Frodo and Sam's entire route in the Middle-earth server? Well, it is, and we did it. If you want to help us, please join our discord server. Please be aware that this project is a Minecraft Parody set in the Middle-earth universe and all rights are reserved under Tolkien domain. You can find both licenses at our github repository. Current state of the modĪs of now, the mod is in Alpha development stage, meaning this project is still a prototype and it's missing many core features we want to add.Īrtistic elements such as textures, models, audio, and more are subject to licensing under either the CC-BY-NC 4.0 license or ARR, because multiple artists worked on it or/and gave permission to use their assets.įor the code, it's under MIT license. To enter the Middle-earth dimension, you will need to right click the starlight phial item. To enter the Middle-earth, you need to use a starlight phial. You'll find a brand new dimension with custom blocks, items, entity, generation, etc. This mod is about the famous universe of the Middle Earth, from J. Welcome to this open-source project of Middle-earth mod. At present, nitrocellulose (NC) and polyvinylidene fluoride (PVDF) membrane are the two solid phase carriers widely used to bind proteins in WB. However, whether IB or LB, the sensitivity of detection of protein or glycans is closely related to the choice of solid phase carrier materials in the process of WB 12, 13. The changes in glycosylation determine the interaction between glycosylation and lectins on the surface of cancer cells 11. For example, the number of terminals sialylated glycans increased significantly in various cancers 8, 9, and the level of core fucosylated glycans also rises obviously 10. It has been reported that during carcinogenic transformation in mammals, severe changes in normal glycosylation occurred. LB is commonly used for detection of protein glycosylation based on lectin–glycan interaction 7. IB is widely used to detect and semi-quantify antigens, determine the relative molecular weight of polypeptide chains and the extraction efficiency of target antigens 6. The process can be described simply as first separating natural or denatured proteins by gel electrophoresis, then transferring the isolated protein bands on the gel to the solid phase supporting material, followed by immunoblotting (IB) and lectin blotting (LB), respectively. Since its inception in 1979, Western blotting (WB) has become a routine experimental method for the determination of proteins and their expression levels in molecular biology and proteomics, as well as used to study protein abundance, kinase activity, cellular localization, protein–protein interaction or monitor protein post-translational modification 5. Therefore, the detection of target protein and changes of abnormal glycosylation are extremely vital for the exploration of new biomarkers for certain diseases. In addition, alpha-1-acid glycoprotein (AGP) as a protein can be used as a prognostic marker of lung cancer 3, and a diagnostic marker for laryngeal cancer 4. Human epidermal growth factor receptor 2 (HER2) is an important indicator for molecular typing of breast cancer, and glycoantigen 19-9 (CA19-9) is used as an important indicator to judge the prognosis of pancreatic cancer 2. What’s more, with the development of proteomics and glycobiology, some proteins and glycoproteins in peripheral blood have become routine tests for diagnosing diseases and predicting prognosis in clinic, such as Alpha-fetoprotein (AFP), a core fucosylated protein used in the diagnosis of liver cancer. Glycoproteins play an important role in cell signaling, immune recognition and cell–cell interaction 1. More than half of the proteins in eukaryotic cells are glycoproteins. Glycosylation is a common post-translational modification of proteins. Protein is the basis of life, which is not only the key component of cell, but also the main executor of biological functions. Bound proteins were stained with direct blue-71, and the staining intensity was quantitated by scanning and densitometry. Moreover, we verified the above results by directly incubating proteins having different molecular weights onto PVDF or NC membranes. The antibody re-probed ability of the two membranes was also explored. Here, by incubating proteins which were transferred to PVDF or NC membranes with a series of antibodies and different types of lectins, we investigated the relationship between the binding ability of these two membranes to proteins or glycoproteins and the molecular weight of the target protein. Which membrane of these two is more suitable for WB has not been reported so far. The most common used membranes are polyvinylidene fluoride (PVDF) and nitrocellulose (NC) membranes. The selection of electroblotted membrane is one of the key factors affecting the detection sensitivity of the protein which is transferred from gel to membrane in WB. Western blotting (WB) is one of the most widely used techniques to identify proteins as well as post translational modifications of proteins. |
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