In Gecko 9.0 or later, this attribute can be set to true if the frame is allowed to be placed into full screen mode by calling its element.mozRequestFullScreen() method. mozallowfullscreen Use allowfullscreen instead. marginwidth HTML 4 only The amount of space in pixels between the frame's content and its left and right margins. marginheight HTML 4 only The amount of space in pixels between the frame's content and its top and bottom margins. Due to widespread misuse, this is not helpful for non-visual browsers. longdesc HTML 4 only A URI of a long description of the frame. height Indicates the height of the frame HTML5 in CSS pixels, or HTML 4.01 in pixels or as a percentage. The value 0 tells the browser not to draw a border between this frame and other frames. frameborder HTML 4 only The value 1 (the default) tells the browser to draw a border between this frame and every other frame. allowpaymentrequest This attribute can be set to true if the frame should be allowed to invoke the Payment Request API. If this isn't set, the element can't be placed into full screen mode. allowfullscreen This attribute can be set to true if the frame is allowed to be placed into full screen mode by calling its Element.requestFullscreen() method. align Deprecated since HTML4.01, Obsolete since HTML5 The alignment of this element with respect to the surrounding context. This element includes the global attributes. None, both the starting and ending tag are mandatory.Īny element that accepts embedded content. Content categoriesįlow content, phrasing content, embedded content, interactive content, palpable content. The top-level browsing context (which has no parent) is typically the browser window. The browsing context that contains the embedded content is called the parent browsing context. Each browsing context has its own session history and active document. However, an can be used within a normal document body. In HTML 4.01, a document may contain a head and a body or a head and a frameset, but not both a body and a frameset. The HTML element represents a nested browsing context, effectively embedding another HTML page into the current page.
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Remember that to make the passive voice, we make the object of the sentence into the subject, add in the correct form of the verb to be, and change the verb to the past participle. The cupboard was moved out of the room by him.ĩ. In this active and passive voice quiz you are given 10 active voice sentences and you have to change them into the passive voice. The pretty dress was being hidden by her.Ĩ. Let’s reinforce this topic by taking a look at the Passive Voice and Active Voice examples below: I made a salad for dinner. We then discussed different types of voices.Ĭonvert the given sentences into passive voiceĨ. Example sentences along with answers have been illustrated. The action's performer may or may not be named later in the text when utilising the passive voice. The subject of the sentence is the one who receives the action, which was highlighted by the passive voice. By making it plain to the reader who is acting in the sentence, an active voice can help assure intelligibility. New workers were hired by the company last year. (Passive Voice) A brownie is being baked by Michael. (Active Voice) The salad was made for dinner. The sentence gets flipped, and the subject is now being acted upon by the verb. Let’s reinforce this topic by taking a look at the Passive Voice and Active Voice examples below: I made a salad for dinner. is, is being, was, will be) + the past participle form of the verb (e.g. In passive voice, the verb is composed of a form of to be (e.g. A main verb is passive when the subject of the sentence is the receiver of the action. In the passive voice, the action’s target, ball, is positioned first as the focus of the sentence. A main verb is active when the subject of the sentence is the doer (or the agent) of the action. This is a simple, direct example of the active voice. The active voice sentence subject (watching a framed, mobile world) performs the action of reminding the speaker of something. Notice how the subject, dog, is performing the action, chase, on the target of the action, ball. Sample active voice sentence with the subject performing the action described by the verb. While passive voice can assist avoid needless repetition, active voice frequently increases intelligibility. This is an example of a sentence using the active voice. In this article example sentences along with answers have been illustrated. There are two types which are active and passive. Voices denote how information is presented. Here ‘him’ is the indirect object, and ‘a book’ is the direct object.In this chapter, we discussed the concept of parts of voices. “ I gave him a book.” - in this sentence, there are two objects - i) ‘him’, ii) ‘a book’. Rule 6: How to change the voice of a sentence with a Direct Object and an Indirect Object. These phrases are used as a single word (verb). Some definite prepositions Laugh at, aim at, arrive at, surprise at, look for, search for, go to, come to, please with, acquaint with, supply with, cope with, sympathize with, prefer to, put down. Passive – The plants are taken care of by my sister. Passive – The stoppage was arrived at by them at 10 o’clock./ The stoppage was arrived at at 10 o’clock by them.Īctive – My sister takes care of the plants. Passive – His behaviour was surprised at by the teachers.Īctive – They arrived at the stoppage at 10 o’clock. Passive – Subject + be verb + main verb (3rd from)+preposition + by + object.Īctive – The teachers surprised at his behaviour. It is impossible for us to tell you what the active voice is without telling you what the passive voice is. (Passive Voice) A beautiful landscape was painted by the artist. (Passive Voice) The cat was chased by the dog. (Active Voice) A new product was launched by the company. (Active Voice) The postman delivers the mail. (Active Voice) The doctor treated the patient. Active – Subject + verb+preposition + object. 100 Examples of Active and Passive Voice Active Voice In the verb, which is one of the most important grammatical topics in English, the roof appears in two different forms as the active voice and the passive voice. (Active Voice) The chef cooked a delicious meal. In a genogram, a square denotes a male and a circle denotes a female.Ī horizontal line connecting the two shows a family. Psychologists use the genogram to help independents and couples in treatment and advice. It permits users to prospect numerous association dynamics, analyze the advancing impact, and recognize courses.įor medical executives, genograms can assist them with recognizing medical risk probabilities. Below are the benefits of using genogram. A theoretician, while Aunt Sameera has not chastised her brother for many years, has blood cancer and has a history of dealing with anger issues.Ī genogram is a multi-genesis diagram of an individual’s ancestry and communal tracery. Even if you desire to support your effort straightforwardly by counting only your instantaneous family, you can rapidly demonstrate in your genogram that your uncle Robert and his wife Sameera have two children, they sent their youngest child to boarding school, that Uncle Robert underwent depression, was an alcoholic abuser. For example, a genogram will exemplify how your grandmother's arranged marriage was fixated before being born through associations and how your cousins ended up in another country in the early 1700s. A genogram will not just exhibit you the names of individuals who are members of your family lineage, but how these individuals interconnect with each other and with additionals who are importantly tangled in their lives (clan, serf, instructors, educators, benefactors, grubstakes, live-in nurses). Although, there is composite interaction inside a lineage part, which can only be acknowledged through the utilization of a genogram. What Is It Used For And Do You Need This Diagram?įamily trees are only a trivial portrayal of collective parent and child connections. Genograms hold an abundance of statistics on the ancestries constituted because they authorize you to exemplify not only how members of an ancestry connect but also how they are an outcome of their time, by their conduct, relationship, and furthermore. Ancestries are the complex structure that interrelates with other clique and kindred groups, of which they are a fragment of, and they cannot be fully acknowledged without the extensive factors in which they reside. Genograms permit to swiftly recognize and acknowledge diverse specimens in the patient's ancestry chronicle that may have impacted the patient's present condition.Ī genogram is a depiction of ancestry that comes living. Several genograms additionally incorporate data on disarray sustained in the ancestry, such as alcoholism, depression, diseases, alliances, and living situations. They hold fundamental statistics established in the ancestry of families, such as the name, gender, date of birth, and death of each soul. They accommodate an abundance of statistics on the pedigree constituted. It is used in a diversity of departments, like medication, mental processes, communal work, genealogy, patrimonial analysis, and education. A genogram is a pictorial depiction of a family ancestry that exhibits comprehensive statistics on association amid solitary individuals. It’s a journey of self-discovery and acceptance, where you can express your true self and celebrate the beautiful person you are. In essence, understanding the sissy lifestyle is about embracing your femininity with authenticity, confidence, and a sense of community. Through various activities, self-reflection, and positive affirmations, you can enhance your self-esteem and develop a deeper understanding of your identity. It’s a journey of discovering your true essence and evolving as an individual. The sissy lifestyle encourages self-exploration and personal growth. This supportive network provides a safe space to share experiences, learn from one another, and celebrate the journey of embracing femininity together. Sissies often find support, understanding, and camaraderie among like-minded individuals. One of the beautiful aspects of the sissy lifestyle is the sense of community it fosters. Remember, confidence is not about being perfect it’s about being comfortable in your own skin and embracing every aspect of yourself. Confidence empowers you to express your femininity boldly, whether it’s through your appearance, mannerisms, or even your voice. It’s about owning your identity, being proud of who you are, and radiating self-assurance. Confidence is KeyĬonfidence plays a pivotal role in the sissy lifestyle. It’s not about conforming to societal expectations but rather about finding joy and confidence in being true to oneself. Embracing FemininityĪt its core, the sissy lifestyle encourages individuals to explore and celebrate their femininity openly and without judgment. It’s about self-discovery, self-expression, and, most importantly, self-acceptance. Sissies embrace their feminine side with enthusiasm, celebrating the beauty of womanhood in their own special way. The sissy lifestyle is a unique and empowering way of expressing one’s femininity, transcending traditional gender norms. This exploration can lead to a more profound understanding of oneself and a greater appreciation for the complexities of gender identity. It’s not about imitating stereotypes but rather about understanding the depth and diversity of feminine qualities. Sissy training provides a safe space for individuals to explore and interpret femininity in their unique way. Exploring Femininityįemininity is a vast and diverse concept. Through this journey, many people find a sense of liberation and happiness, as they learn to love and appreciate themselves fully. It allows individuals to build confidence and self-esteem by accepting and embracing all facets of their identity. Building Confidence and Self-EsteemĮngaging in sissy training can be empowering. It’s essential for individuals exploring sissy training to have a supportive environment where they can express themselves without judgment or prejudice. Acceptance and understanding are key in this process. It’s an opportunity to break free from traditional gender expectations and discover the aspects of femininity that resonate with the individual. Sissy training is not about conforming to societal norms instead, it’s about embracing one’s true self. The Importance of Acceptance and Understanding It’s essential to note that sissy training is entirely consensual and is a personal choice made by individuals who want to explore their gender identity and expression. This exploration can involve various activities, such as dressing in feminine attire, learning feminine mannerisms, and sometimes even adopting a new name or identity. It’s a process where individuals, often males, explore and embrace their feminine side. Let’s embark on this empowering journey together.✨ What is Sissy Training?Īt its core, sissy training is a journey of self-discovery and self-expression. Whether you’re curious, apprehensive, or excited about this path, we’re here to provide clear insights and practical advice. In this beginner’s guide, we’ll unravel the basics, dispel myths, and explore the transformative power of embracing the sissy lifestyle. If you’re new to the world of sissy training, you’re in the right place. Welcome to our comprehensive guide on sissy training – an empowering journey towards self-discovery and confidence. Yet both the Express and the Extreme are rather tiny by traditional router standards. It measures 6.5 inches (16.5 centimeters) square and 1.3 inches (3.3 centimeters) thick. It also has multiple antennas, meaning it can send and receive data simultaneously for faster transfer.Īs such, the Extreme is a bit larger than the Express. The Extreme has other features that make it valuable to power users, including not one but three LAN (local area networking) ports. Those increased speeds, of course, make it a lot easier to send the image- and sound-laden multimedia files that have become staples of both personal and business correspondence. The current unit substitutes an 802.11n chip for the older 802.11g, meaning it's roughly five times faster. With that increase in capacity, the Extreme was suited for small- and medium-sized organizations of all types.Īpple released an update to the Extreme in 2007. It featured all of the same capabilities of the Express, but instead of maxing out at 10 users, it supported up to 50. The company unveiled the first Extreme in 2003, and as with the Express, it used an 802.11g chip. For power users, Apple also unleashed the Airport Extreme, which takes the Express's concepts and beefs them up significantly. The Express isn't the only AirPort in town. And if you're on a Mac, built-in Bonjour capabilities mean users on the network can easily find one another to share pictures, videos or any other kind of data. Plug it into a printer via the USB cable, and presto, any of those 10 users is able to print documents wirelessly. Security-wise, you can set the Express to use WPA (Wi-Fi Protected Access) or WPA2, 40-bit or 128-bit Wireless Security (WEP) and MAC address filtering.Ĭonnect AirPort to your DSL (direct subscriber line) or cable modem, or your Ethernet network, and it provides wireless Internet access for up to 10 users. It's also compatible with both Mac and Windows operating systems. Its small form factor means the Express slips neatly into the side pocket of even a svelte laptop carrying case. If you can't restart using AirPort Utility, just unplug the base station for a few seconds, then plug it back in.That's a lot of features for a device that's only 3.7 inches (9.4 centimeters) high by 2.9 inches (7.5 centimeters) wide and 1.1 inches (2.8 centimeters) thick. On iOS devices, tap the base station in AirPort Utility, then tap Edit > Advanced > Restart Base Station. On Mac, choose Base Station > Restart from the menu bar. If you have more than one base station, restart your primary base station last.Įnter the base station password, if prompted. Open AirPort Utility on your Mac, iPhone, or iPad then select your base station. Updating an AirPort base station causes it and its network services to be temporarily unavailable. If your base station is performing a Time Machine backup or sharing a hard drive on your network, make sure that none of your devices are currently using that drive. Learn what to do if your Mac isn't connecting to the internet or your iPhone or iPad isn't connecting to the internet. Make sure that your base station and Mac or iOS device (iPhone, iPad, or iPod touch) are connected to the internet. Here's how to make sure that your base station is up to date. Use AirPort Utility to update an AirPort base station, including AirPort Time Capsule, AirPort Extreme, or AirPort Express Base Station.Īpple periodically offers software updates (called firmware updates) that improve AirPort performance, security, and functionality. Update the firmware on your AirPort base station You can also download all tilesets and characters at once in the archive " XP Graphics for MV".įree for commercial and non-commercial use in any RPG Maker engine, as long as you legally own RPG Maker XP. The zip archive " XP Characters for MV" below contains all XP character graphics ready for use in MV (193 sheets, most of them single characters). There are duplicate tiles between the sheets if you want to use your tilesets more efficiently, you will need to rearrange some tiles yourself (or wait until I find the time to do it ). XP_to_MV_017-MineTown01 a_PixelScaled.png and XP_to_MV_017-MineTown01 b_PixelScaled.png, respectively. Some of the original tilesheets are split into two MV tilesheets those are named e.g. the 50 XP tilesheets reformatted for MV they can be used in any of the B-E slots. two sets of A2 autotiles (inside and outside) The zip archive " XP Tilesets for MV" below contains: If you don't like the resized tiles and would rather use the 32x32 pixel version of these tiles in MV (with a plugin to change the tilesize), you can find them in the VX Ace thread here. These tiles are meant more as a base for you to work with and less as plug-and-play graphics, although they are ready for use in the engine. (Please note that you must own RPG Maker XP in order to use these tiles.)Īs you can probably tell from the picture above, the result is by no means perfect - especially the autotiles have some issues - and I won't have time to fix them in the near future. As of now, I only upscaled the graphics using the "Fast" method of the RTP Scaler by RyanBram (which can be found here) and rearranged the tiles so that they can be used in RMMV. I resized and reformatted the tilesets from RPG Maker XP for use in MV a while ago and thought I might as well share them here. (Download links at the bottom of the post) You can include motivational quotes such as “never give up”. Either write their name or their title such as “mom”, “dad”, “husband”, or “wife”. You can also add images from the gallery such as an anchor, skull, dragons, hearts (sometimes used to show true love), or other images.Īdd the names of people you love.Since a tattoo will last forever, you want to make sure that you really like the design that you choose. It is better to wait as you might change your mind.Wait a few days and make sure that it is still the design that you like best.Repeat this process until you have a few letter tattoo designs.Click on the download button and download your tattoo image.Some fonts only have lowercase characters and some don’t have numbers or punctuation. Make sure that the typeface you choose includes all the characters that you want to include in your tat. In the text box on the right, type the text that you want on your tattoo.If you move the mouse over each design you will see an enlarged picture of that font. In the left column, you will see different tattoo lettering fonts.Click on the button above to open the tattoo text generator.Tattoo Font Generator How to Use the Tattoo Font Generator Choose a font that has special characters and ligatures if you want to include punctuation. If you include numbers and letters then you will probably want to use the same font for both. If you are going to include numbers or dashes in your tatt then you will need a number font. This font is great for word tattoos with a typewriter style font. See Gothic Font Typewriter font tattoo style Old English fonts are quite popular on tattoos. This old English Roman numeral tattoo font generator will produce Gothic letters or a vintage tattoo font. It is based on a varied and fluid stroke similar to handwriting. A tattoo script font is written in calligraphy and mimics cursive handwriting. The script tattoo is one of the most popular tattoos in the world. Tattoo Font Generator Different fonts for tattoos Try a few of our tattoo lettering styles before you make a decision.Ĭlick on the button below to open our free tattoo font maker and type the text that you want on your tattoo. You want to find a design that you love since it will be on your skin forever. When it comes to tattoo writing, there are no rules. If you show them a sample of the design that you want then it will be easier to explain what you are looking for. When you go to your tattoo artist, show him or her the font style that you would like and see if they can create it for you. You can play around with our free tattoo generator until you find a design that you like. You will also need to decide if you want to use lowercase letters or uppercase letters or a combination of them. It can be a single word, a phrase, or a sentence. You will need to find a tattoo script font you like and that looks good with the image that you want (if you want one) and the style that you like. Tattoo lettering is one of the most important parts of your tattoo if you are looking for a script tattoo. There’s a loose spontaneity to Wyatt and Billy’s sojourn borne out in the style of its documenting, an experimental aesthetic that mirrors the up-for-whatever mindset of its subjects. View image in fullscreen Dennis Hopper and Peter Fonda star in Easy Rider. Hopper used the personal agency that production company Raybert would later allow him to adopt a film-making method befitting the credo he espouses in the film – the open road, no plan and endless possibility. Easy Rider hails from a time when abrasive guys with a lot of vision were given the economic latitude to run the show, or at least their own little slice of it. To him, it was good riddance he’d die before an artist such as himself would take orders from a bunch of pencil-necks in three-piece suits. Roger Corman was originally in talks to produce Easy Rider, and only split ties with the project after Hopper’s foul mouth and erratic behavior drove away a group of potential backers in one pitch meeting. Hopper had a reputation as an uncooperative collaborator, prone to delaying productions and arguing with directors while pursuing his inscrutable creative whims. As much as it ended an era with the flaming crash at its conclusion, the film also launched a new era in its industry, one that gave auteurs unprecedented authority over themselves and their work. While those shifting currents of the counterculture have a perceptible trapped-in-amber quality onscreen, somewhat more pronounced to a modern viewer is how Seitz’s notion applies on the other side of the camera. In his essay for the booklet accompanying the Criterion Collection’s home video release, critic Matt Zoller Seitz describes the film as “a freewheeling take on freedom – what it means and what it costs”. View image in fullscreen Jack Nicholson as George and Peter Fonda as Wyatt in Easy Rider. The generous montages depicting the guys astride their hogs, tearing down the highway to the strains of the soundtrack’s many classic-rock cuts, suggest this as their natural state. They balk at every form of organization, whether it be the humdrum suburbia epitomized by the marching-band parade they infiltrate or the bohemian commune they can’t help but depart. Stopping for their first night out, they decide to make camp in the woods instead of using some of their drug money to put themselves up at a motel. They wish specifically to exist without that dichotomy, to live outside of a close-minded society’s quality judgments about them. They’re no heroes, and aside from the part they play in the international narcotics trade, they’re hardly villains. Their quest follows no imperative higher than the desire to live well and live freely. With a sealed plastic tube full of rolled-up cash stashed in his motorcycle’s fuel tank, Wyatt takes off with Billy for New Orleans in time to catch Mardi Gras. The tacit prologue, scripted solely in untranslated Spanish dialogue and the hurricane roar of a jet engine, follows the pair as they score several bags of high-purity cocaine in Mexico and then sell it to a client (hey there, Phil Spector!) back in Los Angeles. Though Wyatt and Billy’s long hair, sideburns, and far-out couture outwardly align them with the flower children and estrange them from squares at small-town diners giving disapproving looks, they’re far from avatars of peace and love. Situated at the end of its decade, Easy Rider literally and symbolically marks the turning point at which the idealism of the 60s curdled into the indulgent solipsism of the 70s. Roster requirements and scoring for the FFPC Playoff Challenge can be found here. You can also use this as a guide for any fantasy football playoff format, including drafts. To help you build out your squad, I have constructed my NFL Playoff Power Rankings for purposes of the FFPC Playoff Challenge. 2019 Fantasy Football Mock Draft Simulator. This exercise is highly critical in the Fantasy Football Players Championship (FFPC) Playoff Challenge, where you can only choose one player from every team. Fantasy Football Rankings Fantasy Football Cheat Sheet Fantasy Football Stock Report Razzball Trade Analyzer Tool. All rights reserved.When building your fantasy football playoff lineups, the first step is to forecast how far you believe every team can potentially advance. Terms of Use and Privacy Policy and Safety Information / Your California Privacy Rights are applicable to you. You’ve got best ball, head-to-head, weekly, and total points formats. Playoff fantasy football comes in a number of different iterations. ^ Back to Top ^ © 2024 ESPN Internet Ventures. Regular season fantasy football has its fair share of formats and variations, but the overwhelming majority of people play the same game head to head with a 14-week regular season and a three-week playoff. Joyner's players who benefit most from early-season schedule Set your DraftKings fantasy football lineups for Monday here: NFL 2. Schefter's Friends and Family Cheat SheetĬockcroft's quarterback-streaming planner Previously featured in the cheat sheet:Ĭlay's players likely to score more touchdownsīowen's second-year WR breakout candidatesĬlay's players likely to score fewer touchdownsĬlay's toughest, easiest schedules for wide receiversĬlay's ranking of the No. 2023 TOP 200 Fantasy Football Rankings, TOP 200 PPR Cheatsheets TOP 200 PPR Draft / Draft Rankings Powered by If Login button does not work, logoff here, and log back in to fix it. Looking for more details about the players listed in our cheat sheet? Follow these links for the articles containing these picks and a selection of other content that will help you be the most prepared at your draft: Featured in the cheat sheetĬlay's ultimate fantasy football draft board Here it is: ESPN+'s (printable!) fantasy football draft cheat sheet for the 2019 season. This file will be updated regularly from now through the start of the season, so you'll have the latest info and analysis no matter when you draft - and we'll keep adding content as it becomes available. Cockcroft's quarterback-streaming planner So its a mix of per-game production and the number of projected games for a given team. This list is geared toward playoff leagues with PPR fantasy scoring (4/6, 20/10) and that draft only once before the playoffs and count all scoring through the Super Bowl. Mike Clay's running back handcuff rankings This article is part of our NFL Playoff Rankings series. Matt Bowen's rookie running back rankings Delivered straight to your inbox, 100 free. This sheet features 300 players in order of overall draft value, with positional rank, auction value and bye-week information for leagues that reward each catch with a point. 2019 NFL Draft Guide: The Ultimate Cheat Sheet Fantasy. Matt Bowen's Eight favorite players to draftĪdam Schefter's Friends and Family Cheat Sheet A quick, easy-to-use, all-in-one cheat sheet that will help show you which players to draft and when in your fantasy football leagues. 28įield Yates' second-year breakout players Karabell's tiered RB and WR rankings, updated Aug. Our two-page PDF aggregates the names and rankings featured in the following content:Įric Karabell's "Do Not Draft" and "Do Draft" lists In order to help you prepare for your drafts as quickly as possible, we provide you with the ESPN+ cheat sheet, which brings together all of the best analysis from fantasy experts to help win your league. ESPN+ Cheat Sheet Get all of our best ESPN+ intel in one printable cheat sheet to bring with you to your fantasy football drafts. Updated and expanded ESPN+ fantasy football cheat sheetĮSPN has some of the brightest minds in fantasy football, but it can be tough to read every article we publish all summer. A collection of downloadable, printable cheat sheets for the 2019 fantasy football season, including PPR, non-PPR and dynasty/keeper leagues. You have reached a degraded version of because you're using an unsupported version of Internet Explorer.įor a complete experience, please upgrade or use a supported browser Species source of the secondary antibody: there is usually no predictable connection between species source and the quality of the secondary antibody. If the type of the primary antibody is not clear, IgG against the corresponding species can be used. If the primary monoclonal antibody is of a certain subclass of mouse IgG (IgG1, IgG2a, IgG2b, or IgG3), then almost all anti-mouse IgG can bind to it, or the secondary antibody can be selected to specifically target this subclass. If the primary antibody is mouse IgM, then the corresponding secondary antibody should be anti-mouse IgM. Polyclonal antibodies are mainly IgG immunoglobulins, so the corresponding secondary antibodies are anti-IgG antibodies. This is usually applicable for monoclonal antibodies. Type of primary antibody: the secondary antibody must match the class or subclass of the primary antibody. For example, if the primary antibody is a mouse-derived monoclonal antibody, an anti-mouse secondary antibody (goat anti-mouse or rabbit anti-mouse) should be selected. Species source of the primary antibody: the reactivity of the secondary antibody should be consistent with the species source of the primary antibody used. When selecting a secondary antibody, both the type of primary antibody and the requirements of subsequent detection schemes should be considered comprehensively: Usually, there are multiple secondary antibodies suitable for the subsequent detection of the target protein, and the selection can be optimized in specific experiments. If the results are highly correlated, then the antibody is validated for WB analysis. These methods include detection of whether the signal is eliminated or significantly reduced after genetic knockout or knockdown of the target gene analysis of the correlation between WB signals and signals of other detection methods (e.g., MS) in a set of different samples with variable expression of the target protein analysis of the correlation of protein levels by using two or more independent antibodies targeting different epitopes of the same protein expression of the target protein with a tag, and analysis of the correlation between antibody labeling and the detection of the tag. For unvalidated antibodies, there are suggested methodologies to validate the selectivity of the antibodies. There are currently databases that can be used for choosing characterized antibodies with high selectivities, such as Antibodypedia ( ), the Human Protein Atlas ( ), and the Antibody Registry ( ). The selectivity of antibodies directly affects WB results, and poor selectivity may lead to the misinterpretation of the results. This guide, therefore, aims to provide an updated and more concise and useable reference for future experiments and paper writing. Here, we will focus on some essential caveats during the WB experiment. However, concerns about WB have been voiced by many scientific journals in an effort to reduce potential mistakes and increase reproducibility. Therefore, WB remains the most commonly used methodology in the lab for protein detection. ELISA lacks loading controls, immunofluorescence is an in situ technique and is semiquantitative, while MS is expensive and depends on the experimental technique and conditions. Although there are many new alternative technologies, such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence, and mass spectrometry (MS), they all have their own limitations to some extent. Western blotting (WB) is an antibody-based experimental technique used to detect and quantify target proteins, which are often within a complex mixture extracted from cells or tissue. |
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