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Chora antiparos12/8/2023 ![]() Experiencing the Antiparos NightlifeĪntiparos is a Greek island with a vibrant night scene. Note that you won’t find any accommodation options, and definitely not the luxury Antiparos Hotels here rather than Mother Nature in full blossom. You will be impressed by the sugary beaches, with Livadi being the most popular one. Once the largest nature reserve and refuge, it is now a fascinating archaeological site visited by thousands of tourists every year. This uninhabited minute islet off the Antiparos coast welcomes you to an ancient sanctuary second only to the UNESCO World Heritage site of Delos Island. Speaking of boat tours, you could also book a trip to the mesmerising Despotiko Island that usually departs from the charming village of Agios Georgios. Don’t forget to swim in the emerald waters during a boat tour or try snorkeling. In fact, Antiparos, Greece, counts several secret sea caves waiting to be explored. ![]() However, the Antiparos Cave is not the only spectacular cove on the island. It is the awe that this place inspires nearly all of its visitors year after year! As you go down the 411 steps leading to the heart of the cave, you will notice a chilling sensation running up and down your spine. Home to a beautiful chapel (Agios Ioannis Church) and majestic stalagmites and stalactites, the Cave is around 85m deep and highlights a massive 45 million-year-old column. Hands down, the Cave is the top attraction of Antiparos, Greece. Or you could choose to stay in the comfort of your Hotel in Antiparos and share romantic moments with your significant other. Although you will need to walk an incredibly scenic path to get there, the experience is definitely worth it. Overlooking Sifnos island, the lovely stretch of sand is the perfect and most romantic place to kiss the day goodbye. One of the most popular spots to watch the sun glide behind the horizon is Sifneiko Beach (aka Sunset Beach). Note: Most Antiparos hotels are actually built near or around the Castle, offering guests incredible panoramas that soothe the eye and ease the soul. Do check out the gorgeous Gothic gateway near the main tower and do not leave without treating yourself to the heart-stopping views of the archipelago. Built to help the islanders protect themselves from invaders and pirates, it now stands a proud reminder of its once glorious days. Though not a full-scale castle, it still is a characteristic sight of the island dating back to 1440. There, you will find plenty of cafes, tavernas, restaurants, seaside bars, and shops selling from souvenirs to jewelry.Īs you wander around, chances are the road will take you to the imposing Antiparos Venetian Castle. Antiparos, Greece, as every Cycladic island, gathers all of its vibrant nightlife and attractions in its capital (aka Chora or Antiparos Town). You will soon realise that the more you mingle with the locals and stroll the cobbled streets, the more interesting opportunities for more things to do open up. This guide will give you a small taste of the very essence that makes Antiparos such an extraordinary holiday destination. Without a doubt, this authentic Greek island seems to have been created to please everybody, from couples and groups of friends to solo globetrotters and families with kids. ![]() No wonder many Antiparos hotels are an attraction on their own, impressing with their style, chicness, amenities, and comforts. This beautiful dot in the sea is home to pristine villas owned by celebs the magnitude of Tom Hanks! Featuring whitewashed houses with bougainvillea-filled balconies, cubic-shaped constructions, and blue-domed churches, all distinguishing of traditional Cycladic architecture, Antiparos is a sight for sore eyes, indeed. Despite its relatively small size, Antiparos, Greece remains a top destination in the Aegean Sea among people searching for a slice of heaven on earth. ![]()
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2017 escape12/8/2023 ![]() ![]() ![]() Your Ford Escape may have a lower towing capacity based on the drivetrain, axle ratio, and if your vehicle has a towing package installed. Note that these are the max towing limits for each 2017 Ford Escape engine. The 2017 Ford Escape comes standard with front-wheel drive, with all-wheel drive optional on most trims.Ģ017 Ford Escape FWD Tow Capacity: 3,500 poundsĢ017 Ford Escape AWD Tow Capacity: 3,500 pounds There are three engine options for the 2017 Escape, which are a 2.5L I4 engine that produces 168 horsepower and 170 pound-feet of torque, a 1.5L EcoBoost I4 engine that produces 179 horsepower and 177 pound-feet of torque, and a 2.0L EcoBoost I4 engine that produces 245 horsepower and 275 pound-feet of torque.Ģ017 Ford Escape 2.5L I4 Engine Towing Capacity: 1,500 poundsĢ017 Ford Escape 1.5L EcoBoost I4 Engine Towing Capacity: 2,000 poundsĢ017 Ford Escape 2.0L EcoBoost I4 Engine Towing Capacity: 3,500 pounds 2017 Ford Escape Drivetrain Towing Specs Here’s the max tow capacity broken down for each package on the 2017 Ford Escape: 2017 Ford Escape Trim LevelĢ017 Ford Escape Towing Capacity by Trim Level Chart Towing Capacity By Engine Size & Drivetrain 2017 Ford Escape Engine Towing Specs Some engines and features are not available on certain configurations, so they can each have different max towing limits. There are many trim levels for the 2017 Escape. 2017 Escape Towing Capacity By Trim Level You can also read our guide on what makes a vehicle towing capacity to learn all the aspects that go into it. You can check out our guide to Ford Escape towing capacity by year if you’re looking at a different model year’s max capacity or if you want to compare different years of this vehicle. ![]()
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Strike pack xbox one series x12/8/2023 ![]() ![]() Another new movement option, is the Ledge Hang, which this allows the player peek from a Mantle and look around. The new movement options introduced in Call of Duty: Modern Warfare, like Tactical Sprint and Mounting are back, and now with a new one added, the Dive to Prone, returning from past games, that works together with Sliding. On the Ground War maps, there are also water vehicles that will allowed a better mobility. Different of Call of Duty: Modern Warfare, now the player will be able to swim. Different vehicles are available for use on the Ground War maps.Ĭall of Duty: Modern Warfare II also brings the possibility of Swimming and Aquatic Combat. Now, the player can destroy vehicle sections, like bumpers, doors and Blowing out tires. The game also brings a realistic combat in the vehicles. Kills, assists, objective and tactical plays can unlock these more quickly. This consists of four Perks, two base, plus two more known as a “Bonus” and “Ultimate.” All are earned during a Multiplayer match. The Perks system changed compared to past games. This allows the player to unlock new weapons and have attachments shared between weapons of a platform and universal attachments that will be shared for all weapons in the game. Now weapons are grouped into “Platforms”, allowing for complete control over the weaponry that the player bring into matches. ![]() One of the big changes is the complete revamp in the Gunsmith. The Multiplayer of Call of Duty: Modern Warfare II brings many changes compared the previous Call of Duty: Modern Warfare Multiplayer. Multiplayer " Infinity Ward brings fans state-of-the-art gameplay, with all-new gun handling, a new Gunsmith, and a suite of other gameplay and graphical innovations that elevate the franchise to new heights." - Description
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Rookie dynasty superflex rankings12/8/2023 ![]() ![]() But obviously, he is crowded by Ken Walker, too. Again, I don't really see the upside outside of the tight ends and maybe Charbonnet, as well. Laporta's a little bit crowded, but still, that's going to be a really good offense. So influx of talent, I think some good landing spots as well for Kincaid, for May. Like, that was the state of the position. JAY CROUCHER: Yeah, and no offense to him, but the fact that we were talking about Juwan Johnson as a top 10 tight end and Jason Johnson, like his over unders for receiving yards was set at like. I mean, three tight ends in the top 20 is just good time for a position that's starving for help. We saw Kincaid at number eight, there, but Jay, right here. All of these ranks will be available and, .ĬONNOR ROGERS: I think one of the really good things is, we get to 11 through 20, resurgence at tight end that we've talked about. It's Bryce Young and CJ Stroud towards the end of my top 10. So there you go.Īnd then you get into Zay Flowers, we mentioned Dalton Kincaid. We're not talking about a Justin Jefferson, here. And as much as we like Jordan Addison or Quinton Johnson, the fact is, top 20 wide receiver is likely- we're not talking about a Ja'Marr Chase, here. So there's definitely a chance that within three or four years, we're talking about Anthony Richardson is the number one quarterback in fantasy. He could be a guy with that kind of upside with the offensive line, and with Pittman if they add another piece, there. MATTHEW BERRY: He could be Josh Allen, exactly right. ![]() Like, he could be Cam Newton and Justin Fields My argument for Anthony Richardson at four, even though it's a one quarterback league, it's hard to trade quarterbacks and quarterbacks have is that, number one quarterback in fantasy is within the realm of possibility for Anthony Richardson. If you want to take Addison and Johnston over him, I could certainly argue that. But in a one quarterback league, you've got Bijan, then JSN, Jahmyr Gibbs, who we talked about. A lot of dynasty leagues are Superflex, so obviously the rankings would change with Superflex. ![]() so the point here is, you see there in small gray letters, this is for one quarterback dynasty league. I think what happens after that is where things are going to get really, really interesting. Little dynasty action, here.ĬONNOR ROGERS: I think no surprise who sits at the top- Bijan Robinson. Video TranscriptĬONNOR ROGERS: All right, Berry, let's take a look at rookie ranks. Matthew Berry, Connor Rogers and Jay Croucher break down their rookie rankings for one QB and Superflex dynasty leagues, discuss long-term outlooks for the 2023 NFL Draft class and more. ![]()
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Euphoria party activities12/8/2023 ![]() ![]() Play icon The triangle icon that indicates to play 1. And if you're struggling to come up with a presentation topic that people will legit pay attention to, we have you covered with these PowerPoint night ideas. ![]() It’s a super simple way to keep yourselves busy for the night-add wine and make a drinking game out of it if you wish. Prepare your squad and get fancy with your PowerPoint skills/that Canva theme (I know your resume says "proficient in Microsoft Office").or just use WordArt bc who cares. Make it happen virtually using your screen share button, or take turns presenting on your TV screen with the help of an HDMI chord, AirPlay, or Chromecast. But, if you have a lot of friends (love that for you), you can just limit the number of slides, or present in groups. These babies work best with a small-ish friend group, so there aren’t a million different presentations. They're honestly pretty exciting and hilarious. It occurs when friend groups and sorority sisters decide they wanna make presentations (particularly on topics that usually would not get covered in a class). Enter: PowerPoint nights.Ī PowerPoint night is a fun, stay-in-sweats activity that has been blowing up on TikTok for a while. That's why we're here to introduce you to a fabulous ~indoor activity~, so you and your friends can stay in that sweet, sweet AC as long as your hearts desire. The world is low-key burning ( but actually), and there's a solid chance you need a shower after the commute from your front door to your car/subway/bike. Let us all take one step at a time, being respectful of others’ feelings and being ready to help each other, towards a new normality.Welcome to peak summer 2023, aka "holy it's way too hot to go outside" szn. Each person needs to acknowledge that anyone reacts differently to changes, but at the same time most likely somebody else feels the same as we do. The world will not go back directly to what it was in 2019, as we all lived a traumatic period of stress and anxiety. Beginning within a safe and controlled environment, with a few friends or family members, would allow them to keep track of their behavior and risks, and evaluate which situations make them feel comfortable and which need more psychological work and preparation. In order to slowly re-enter a pre-COVID-like social life, people could start taking small steps to fight their anxiety following advice from official sources and starting to push themselves by attending small gatherings first, eventually increasing their confidence. The second, experiencing this peer pressure would find themselves in these uncomfortable situations, which would likely increase their stress disorder, feelings of loneliness and ultimately decrease their interest in being in these crowded situations. The first, feeling like their own point of view is shared by the majority of the population, will try and push the second type into uncomfortable situations. The second perspective is that of those still scared of the virus, those who got used to being on their own throughout the pandemic and now feel fearful of crowded spaces. ![]() These people are looking forward to experiencing fun and activities with a large group of peers again. There are two points of view to consider: the first is that of those tired of being alone, with no social contact. Gatherings can become very stressful situations for some sensitive individuals, those who are now unused to having their personal sphere invaded. ![]() Some people still face mental health issues arising from the fear of getting sick. However, the psychological impact of COVID-19 will not stop just by reopening everything. From the Eurovision Song Contest to Formula 1 races, people seem to be focused on one thing: going back to normality. In the first months of 2021, a wave of positivity brought on by vaccine campaigns and decreasing numbers of hospitalized cases have moved organizations to organize events and activities as they would have, pre-pandemic style. Additionally, the uncertainty of future development, economic issues and changes in family environments created a cascade of negative mental responses: anxiety, depression, post-traumatic stress disorder, and other trauma- and stress-related disorders. The fear of getting sick or spreading the virus to our loved ones increased the level of stress and worry for every individual. Mental health has been a hot topic throughout this last year. Peer support and professional aid are key to restore social confidence one step at a time. If many people are looking forward to going back to normality in the post-pandemic world, then equally many feel anxious in imagining themselves surrounded by people. ![]()
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Inet pro network scanner12/7/2023 ![]() ![]() IP addresses, and DNS names with monitoring tools like MAC addresses and a DNS. * Try it out with our FREE version of IP Scanner available in the App Store for macOS & iOS Still not sure if IP Scanner Ultra does what you need? * Or contact us directly with any questions at - we always love hearing from our customers.INet - explore your network! With iNet you know exactly what's going on! Find out about security risks and keep your network under control. This easy to use App outlines a computer network even to the unexperienced user.Did you ever wanted to know which devices are connected to a network and which services are running? Are there ports opened for communication with the outside world? Find out with iNet network scanner. You will get a profound answer.iNet shows the devices connected to your local network, for instance computers or routers, along with their name, IP and vendor. Most Apple devices are displayed with their correct icon. iNet scans on a very low level and shows even hidden devices.All running Bonjour services are shown along with their real name.iNet stores the last ten scans automatically. With the additional "save scan" feature you can save performed scans, name them and display them later for comparisons.If you want to know what's going on in you computer network - iNet is for you.P.s.: iNet is the little brother of iNet Pro. This free version of iNet offers the same sophisticated network scan engine as the Pro version. #INET NETWORK SCANNER MAC UPDATE#Īnd you can easily update to iNet Pro from within iNet if you want to use its full feature set.P.p.s.: The free version of iNet is free of nasty advertisings. MAC iNet Network Scanner v2.8.1 macOS ENG. ![]() You will never be bothered by popups or slide-ins that propagate other Apps. INet utilissimo strumento per il controllo della vostra rete. #INET NETWORK SCANNER MAC PROFESSIONAL#. ![]()
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Crumb bakery12/7/2023 ![]() ![]() carries the genuine warmth and personal touch that sets it apart. Our commitment to handcrafted excellence ensures that every treat from crumb. Way down in South Philly, Dodo Bakery makes freshly baked Hong Kong-style pastries and buns, plus bubble tea and smoothies. In a world dominated by mass production, we proudly stand apart by infusing each and every dessert we make with abundant love and passion. A post shared by Dodo Bakery (thedodobakery) Dodo Bakery, South Philly. We have a simple goal: to reignite those heartwarming emotions within you. From the tantalizing aroma that fills the air to the irresistible flavors that dance on your taste buds, our ultimate aim is to create treats that satisfy your cravings and nurture your soul. Welcome to The Perfect Crumb Bakery We carry a variety of cookies, muffins, breads, pies, cakes, cupcakes, and more. Each recipe is thoughtfully perfected, ensuring every bite captures the essence of homemade goodness. We believe that desserts hold a special place in celebrations and strive to contribute to the memories you cherish.Īt crumb., we are dedicated to crafting baked goods of exceptional quality that evoke a sense of comfort and familiarity. Pumpkin, apple, pecan, mixed berry crumb, traditional cheesecake, salted caramel and pumpkin cheesecake are the offerings for Thanksgiving. As a homegrown company, we understand the profound impact of nostalgia and seek to recreate those heartwarming moments through our delectable creations. This scrumptious breakfast treat is lightly-sweetened with organic coconut sugar and available in a variety of wonderful flavors, including lemon blueberry. ![]() You can find Crumb and Sponge products a number of places: local pickup here in SE Mequon (we also deliver!), Ozaukee REKO Ring, West Bend REKO Ring, DreamPort Market Store in Port Washington, as well as some local area farmer's markets in the summer and winter.Welcome to crumb., where our passion for artisan-style baked goods shines through in every delightful bite. Our artisan bread is handmade using a traditional approach with overnight proofing and loaves formed by hand, creating delicious. ![]() Hopefully by now you are wondering where to find us. Known for our Freshness and Imagination, our Delicious Cookies are bursting with Fun-Filled Flavors. That's because I couldn't do this without the unwavering support of my husband and children - they are my strongest supporters and also my first taste testers! I'm the baker behind it all, but you'll notice the website is written as "we". We will continue to work on custom cake, iced cookie and. Smith Best restaurant in Washington or Charlotte counties. MenuPix User asked the girl if she’d come to my table to take my order, she just said oh i didn’t see you, then never takes my order she’s so annoying. We do this by using quality ingredients, such as unbromated / organic flours, organic milk, organic eggs, European style butter, and not using any preservatives or additives. The Cookie & Crumb no longer has regular retail hours and is now a custom/pre-order bake shop. I promise satisfaction, not just because it is good business, but because chances are, like my father, you work hard for your money and. Top Reviews of Crumbs Cafe & Bake Shoppe. Our mission is easy to understand: feeding yourself and your loved ones food that you can feel good about, and that leave you feeling good. Being a bread baker for over 20 years, and already well in love with making sourdough for several years at that point, finding the the focus of my micro-bakery was probably the easiest thing about the pandemic! Just like that, the concept for Crumb and Sponge was baked: "scratch baking" with high quality ingredients, available for others to share with their own loved ones. With encouragement from family and friends, I realized that there were a lot of other people interested in artisan baked goods, made with high quality ingredients and without any preservatives or additives. ![]() Back in 2020, in the midst of the pandemic, as our home transitioned from just our home to a dual office for my husband and I, a school and let's face it, gymnasium for my two young children, my stress baking went beyond what my loved ones could consume. ![]()
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Draftsight 3d zeichnen12/7/2023 ![]() ![]() ![]() If I cant open this program and draw something that I have previously drawn on other software packages within relative ease then I'm at a loss. In my opinion 2D CAD software is 2D CAD software, the only deciding factor is the price (that's pretty much the only difference between AutoCAD and DraftSight). This is probably the reason why they recommend to purchase the training document alongside this software. For me, I can draw something on DraftSight in half the time. The ease of use is quite terrible and it unfortunately effects the efficiency. I literally spend most of the time on this software trying to figure out how to trim or get a polyline to bend or some other simple task that shouldn't take long at all. You have to do everything separately through a series of different tool editors within each tool. This software does not operate like that. When I want to trim a line that over laps another line, I want to select the trim option then trim the line I want to trim, that's it! From my experience so far the auto trim is simply useless. It might be because I'm swapping over from different software after so long, but some of the simplest tools such as trimming seem to be overly complicated. This could be used to create some really complicated and in depth drawings. It seems to be optimized well and I haven't found any bugs as of yet. It looks professional and has a lot of features. I currently use DraftSight, however this is half the price for essentially the same thing. The best thing about this software is the price. To be honest it costs more because at the moment it's a better software. Experiences cost-effective 3D design tools for CNC machining and 3D printing. Reduces time on 2D Drafting and documentation with the powerful productivity tools. Provides flexible offerings to suit different business and budget needs. Unfortunately I will be sticking with DraftSight for now despite costing double the price. DraftSight Benefits: Allows users to create, edit, view, and mark up any type of 2D and 3D DWG file. I use that on a daily basis and it's extremely easy to use and is centered around the same mechanics as 2D CAD software. Vcarve (which is a vector CNC software) is a great example of what I mean. Simplifying some of the standard tools in my opinion would be helpful. ![]() However updates / improvements are needed in my opinion. Ideal for organisations with multiple sites, designers, makers, manufacturers, and professionals.Kommentare: This software has potential to be amazing, especially if it stays at the same price. If you have multiple users of 2D CAD, then DraftSight Enterprise should be a serious consideration. Accredited online, onsite and classroom training.Local support (award winning support from Solid Solutions).Network License (ideal for multiple users).Dynamic Blocks, PowerTrim, DrawCompare, and automatic layer assignment.All licenses include API access and productivity tools.For organisations who need the power of DraftSight Professional or Premium plus network licensing then our Enterprise license allows you to float your 2D CAD licenses to maximise efficiencies and reduce your cost of ownership. Its familiar user interface facilitates a quick transition from other CAD applications like AutoCAD.ĭraftSight is available as a perpetual licence and comes with award-winning support from our technical team. DraftSight allows you to do more with a 2D drafting and 3D design experience that lets you create, edit, view and markup any kind of 2D and 3D DWG file design. ![]()
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F150 cobb tuner12/7/2023 ![]() ![]() All throttle closures are not necessarily bad. Adjusting the Wastegate Position Base table is the best way to help reduce throttle closures. The most common throttle closures will happen at peak torque when TIP Actual is likely surpassing TIP Desired. We recommend adjusting these limits higher to take full control of the throttle. There are two main offenders that will result in throttle closures: Load and Boost. ![]() The factory tune allows the throttle to close to less than 20* (of the 82* max) which can result in extremely consistent torque delivery. Throttle Closures – The primary method of controlling torque on the Raptor is done with the assistance of the drive-by-wire throttle system.MAP Based – These vehicles use a MAP based airflow system. Watching the Spark Source and HDFX monitors will give you a good idea of which set of tables are being utilized at any given time. The majority of heavy load operation will take place in the borderline timing tables, however this can be clipped by any other timing table from the other categories if they are lower. There are up to 16 primary tables for each method, along with accompanying compensations. The ECU also allows full dynamic advance and retard based on octane learning and knock sensor feedback. Ignition Control – The Raptor consists of three primary methods to control spark: MBT, Borderline, and Cylinder Pressure. Raising wastegate position directly with out modifying other limiters will have little to no impact other than to cause undesirable PID activity and oscillations.įuel Control – The Raptor operates in a constant closed-loop state, constantly utilizing the A/F values in its tables and making closed loop adjustments via the feedback from the factory wide-band O2 sensors (1 per bank). The general strategy of the Raptor is to convert and crosscheck mainly torque, load, and airflow, choosing the lowest value, and then clipping the engine output at that value. Engine torque is calculated based on numerous variables such as: pedal position, air flow mass, knock, KOM, and RPM. Our OTS maps should have good base settings which can be modified further if need beīoost Control – The Raptor does not directly target boost in stock form but rather a target engine torque. The axes are generally index based and may require manipulation to both the index and/or the data to properly display graphs or to utilize greater breakpoint resolution. On tables that would require it, we have broken these out for editing in separate folders. In these tables both the X and Y axis have "paired" data. Here we will go over a few of the basic details and terminologies that are specific to Ford before we begin tuning on a COBB Accessport equipped Ford Ecoboost vehicle.ģD Breakpoint Setup– The Ford ECU uses a new style of 2D axes in select 3D tables. Please contact COBB Support if you have any questions.Introduction Getting to know the Ford F-150 Warning! Installation of this Accessport over anything other than OEM or COBB software may result in ECU/TCU failure. With 165% more surface area over stock, the intake produces more air-flow and a more audible turbo sound when building boost at low RPM. With 165% more surface area over stock, the intake produces more air-flow and a more audible turbo sound when building boost at low RPM.Īlso, we combined the improved airbox design with a high flow, dual inlet, tapered-oval filter. Also, we combined the improved airbox design with a high flow, dual inlet, tapered-oval filter. Powder coated tubing, composite material, and a COBB nameplate transform the engine bay to look as sharp as the iconic exterior. The injection molded - fiber reinforced airbox lid incorporates a closed-cell foam gasket to seal the filter from mud, dirt, and debris. The factory piping from the air-box is replaced with dual 3" mandrel bent aluminum tubing to maximize airflow while custom silicone couplers ensure perfect OEM-like fitment. And what's looks without brains? More processing power, memory, and storage to flash faster while saving all of your maps and logs. ![]() Fully customizable from the look (multiple bezel colors), layout (set up race, street, or dyno gauge clusters), and mounting options (choose from 1,000s of standard vehicle mounts). ![]() Simple ECU flashing with gauge package! The new in-cabin mountable Accessport V3 has a large, full color, higher resolution screen which allows you to monitor up to six parameters in real time. ![]()
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Antibodies in northern blot analysis12/7/2023 ![]() Proteins are commonly separated using polyacrylamide gel electrophoresis (PAGE) to characterize individual proteins in a complex sample or to examine multiple proteins within a single sample. Gel electrophoresis is a technique in which charged molecules, such as protein or DNA, are separated according to physical properties as they are forced through a gel by an electrical current. The indirect method offers many advantages over the direct method, which are described below. Labels (or conjugated molecules) may include biotin, fluorescent probes such as Invitrogen Alexa Flour or DyLight fluorophores, and enzyme conjugates such as horseradish peroxidase (HRP) or alkaline phosphatase (AP). Subsequently, the primary antibody is detected using an enzyme- or fluororophore-conjugated secondary antibody. In the indirect detection method, an unlabeled primary antibody is first used to bind to the antigen. This detection method is not widely used as most researchers prefer the indirect detection method for a variety of reasons. With the direct detection method, an enzyme- or fluorophore-conjugated primary antibody is used to detect the antigen of interest on the blot. One common variation involves direct versus indirect detection. Procedures vary widely for the detection step of a western blot experiment. Whatever system is used, the intensity of the signal should correlate with the abundance of the antigen on the membrane. Fluorescent blotting is a newer technique and is growing in popularity as it affords the potential to multiplex (detect multiple proteins on a single blot). Alternatively, fluorescently tagged antibodies can be used, which require detection using an instrument capable of capturing the fluorescent signal. However, digital imaging instruments based on charge-coupled device (CCD) cameras are becoming popular alternatives to film for capturing chemiluminescent signal. The light output can be captured using film. The most sensitive detection methods use a chemiluminescent substrate that produces light as a byproduct of the reaction with the enzyme conjugated to the antibody. Chromogenic substrates produce a precipitate on the membrane resulting in colorimetric changes visible to the eye. Often the secondary antibody is complexed with an enzyme, which when combined with an appropriate substrate, will produce a detectable signal. ![]() Most commonly, the transferred protein is then probed with a combination of antibodies: one antibody specific to the protein of interest (primary antibody) and another antibody specific to the host species of the primary antibody (secondary antibody). Next, the membrane is blocked to prevent any nonspecific binding of antibodies to the surface of the membrane. Subsequently, the separated molecules are transferred or blotted onto a second matrix, generally a nitrocellulose or polyvinylidene difluoride (PVDF) membrane. The first step in a western blotting procedure is to separate the macromolecules in a sample using gel electrophoresis. ![]() |