Cajun Ribeye 39.59 12 oz ribeye, shrimp, crawfish, tomatoes, lemon butter, green onions. Terrible night, hopefully it was just the evening itself and not the restaurant. Served with a side & your choice of dinner Caesar salad, dinner salad (with a choice of honey-mustard, chunky bleu cheese, ranch, Thousand Island or balsamic vinaigrette), or upgrade to a wedge salad. 502 Riverwalk, San Antonio, TX 78205 +1 21 Website Menu. Not sure why a chain like that would not be properly stocked with steak. 1,548 reviews 56 of 2,380 Restaurants in San Antonio - American Steakhouse Gluten Free Options. 5 'fall-off-the-bone' ribs with grilled chicken. BBQ Ribs and Chicken Family Pack Large 121.00. I wasn't super hungry, so I just had their wedge salad. I am not a big steak eater, but I love that Saltgrass offers a variety of non steak options. Also, apparently the good steaks all went to the large group they were serving because my filet was embarrassing. Choice of two sides (mashed potatoes, green beans, broccoli, or macaroni and cheese), and 1 salad choice (dinner salad with (ranch, balsamic vinaigrette, honey mustard, or bleu cheese) or dinner Caesar). 1400 photos Elite ’22 Went here on Thursday night for a late dinner with some family members and enjoyed myself. While we were eating our steaks, another server showed up with our two original glasses of wine. Finally after literally waiting 45 minutes to get two glasses of wine, I just told the server to bring us a bottle and we then wouldn't have to continue to wait. All Steaks, Seafood & Favorites served with choice of a side (add 90-660 cal) & your choice of Soup or Salad (add 100-380 cal). The server continued to say that the bartender was just behind. We were just trying to relax a bit over dinner. The Artichoke/Spinach Dip and Fried Mushrooms starters went fast. Well the appetizer came and went with no glass of wine. 121 36 Reviewed Apvia mobile A Must with Wedge Salad Four couples, we all were pretty happy with atmosphere, service and especially the food and appetizers. I asked that we wait to order our meals until we had received our appetizer and then could possibly have a second glass of wine with our dinner. We ordered a couple of glasses of wine to go with our appetizer. They were serving a large group, so apparently they couldn't handle anything more. Saltgrass Wedge Salad Serving Size: 1 salad 267 Cal 14 9.4g Carbs 69 21g Fat 17 11.9g Protein Track macros, calories, and more with MyFitnessPal. Served with fries.We have eaten at the Salt Grass restaurants before, but this particular night we had a terrible experience. Served with fries.Īmerican cheese, mac & cheese, bacon. Served with fries.īacon, jack cheese, lettuce, tomato, onions, pickles, honey mustard. Wonderful steak, asparagus, baked potato, and wedge salad food was fantasticHowever, I gave 4 stars instead of five because the restaurant wasn't packed and the customer service was not fully executed here (waiter was very scripted, although very nice, and forgot to provide us with silverware and also did not ring in our daughters side order. 6 oz Top Sirloin and Chicken Family Pack Large. Served with a choice of two sides (mashed potatoes, green beans, broccoli, or macaroni and cheese), and 1 salad choice dinner salad with (ranch, balsamic vinaigrette, honey mustard, or bleu cheese) or dinner Caesar. Garden greens, tortilla strips, bacon, eggs, tomatoes, croutons.Ĭenter-cut Top Sirloin*, crisp iceberg wedge, tomatoes, bacon, blue cheese crumbles, red onions, ranch and balsamic vinaigrette.Īmerican cheese, lettuce, tomato, onions, pickles. 3 center-cut top sirloins with grilled chicken. Garden greens, fried chicken, cheddar cheese, bacon, eggs, tomatoes, croutons. Served with a side & your choice of dinner Caesar salad, dinner salad (with a choice of honey-mustard, chunky bleu cheese, ranch, Thousand Island or balsamic vinaigrette), or upgrade to a wedge salad. Our homemade salad dressings are honey mustard (add 260 cal), chunky blue cheese (add 340 cal), ranch (add 300 cal), Thousand Island (add 320 cal), balsamic vinaigrette (add 380 cal). 12 oz ribeye, shrimp, crawfish, tomatoes, lemon butter, green onions.
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The median income for men was $86,000 per year, but only $57,000 for women. In 2015, median annual salaries of full-time male and female workers in science and engineering revealed a large wage gap. The Persistent Wage Gap in Science and Tech Additionally, the number of women in physical sciences rose from 21% to 28% over the same period. Between 19, the number of women working in engineering increased from 9% to 15%. While STEM is still male-dominated, the proportion of women in STEM has been increasing in recent years. Only 38% of women in STEM who work in male-dominated workspaces believe that they are treated fairly for advancement opportunities. Additionally, many women believe they are not treated equally when it comes to promotion opportunities. Women who do find entry-level work are also paid an average of $4,000 less than male counterparts with the same qualifications.ĭiscrimination in entry-level work makes it even more difficult for women to achieve equal representation in higher levels of STEM, leading to only 18% of leadership positions being held by women. Women are less likely to be selected for entry-level STEM work and are 30% less likely to receive an interview than men with the same experience and qualifications. Part of the reason for this prominent gap is generational gender bias. Gender distribution varies among different jobs in science and tech, but women in engineering are severely underrepresented as only 15% of engineers are women. As stated above, women make up only 28% of science and engineering workers despite being 50.2% of the college-educated workforce. Gender distribution in STEMĮven though women currently make up half of the college-educated workers' population, they are highly underrepresented in STEM. Sign up here and check which scholarships you are eligible to apply for right now. You can click on any of the scholarships above to start applying! While this list is a great place to start your scholarship search, you can find even more exclusive scholarship opportunities on. To help you fund your education and overcome gender obstacles, we’ve created this list of scholarships just for women in STEM. 7% of female graduates in 2016 earned a STEM degree while 15% of male college students did the same.ĭue to the employment gap in STEM education between male and female workers, there are many scholarships available to support women as they pursue degrees and careers in STEM. Women are also less likely to pursue a STEM degree than men due to lack of representation and the stigma that women in STEM usually face. However, high-paying fields such as mechanical engineering and computer science are still overwhelmingly male, with women engineers making up only 9% of mechanical engineers and roughly 20% of civil engineers. Women do make up the majority of certain fields, such as social sciences, and have reached parity in others, such as biology. Women make up only 28% of the science and engineering workforce despite being 50.2% of th e college-educated workforce. STEM jobs in particular, especially the highest-paid STEM fields and positions, are still male-dominated. Women have made vast progress in the academic world, now outnumbering their male peers, yet they are still underrepresented in many fields. Lara's weight pulls it down, activating a mechanism that lowers a square column on the other side of the room. When you're ready to move on, climb the dais and jump to grab the switch. In the Nintendo Wii game, on the wall to the left of the throne, you'll find a RUBBING REWARD (9/12) called "The Harvest." In all versions of the game, there's a box of shotgun ammo on the floor behind the dais. Cross the room to the dais with the golden ankh switch behind it. This tall room has numerous ledges high above. CHECKPOINT 5 registers as you enter the next room. To get past this one, stand close and run through just as the first set of blocks begins to open. Pass through the same way you did before and continue to the double block trap beyond. Continue through a small room to another block trap. Then when they close completely, run toward them, and Lara should pass through safely as they start to open. To get past the block trap ahead, stand a few steps from the stone doors. Instead go through the doorway flanked by Ankh symbols just above the waterline on the opposite side of the pool.įollow the hall to CHECKPOINT 4. There's an alcove with a switch off to one side, but the sturdy bars prevent you from reaching it. Swim across to the other side to find a small medipack behind another column. (This leads back up to the entrance, but don't climb up yet.) Pick up some 50-caliber clips near one of the columns on this side of the pool. Climb out of the water on the ledge with the ladder. This walkthrough describes just one possible sequence.ĭrop off the bridge into the pool below. You don't have to explore the side areas in any particular order. Getting the rest of them will be a little more complicated than this. There are three similar artifacts on the remaining faces of the obelisk. Walk out onto the bridge and take the EYE OF HORUS off the obelisk. Then pull the switch to lower the bridge. Place it in the gear panel and rearrange the gears so the mechanism works again. The missing COG is lying on the floor nearby. Go to the right, pick up a small medipack behind the column nearest the switch and then use the switch to drop one of the four bridges leading from the side areas to the obelisk. At the center of this open area stands a tall, square obelisk. THE OBELISK - EYE OF HORUS SWITCH & ARTIFACT:įrom CHECKPOINT 1 at the base of the ramp, head up to the sunny courtyard. Kills: 7 Supplies: 22 (23)* Other: SEAL OF ANUBIS, EYE OF HORUS, ANKH OF ISIS and SCARAB OF OSIRIS (also 4 COGS - Wii version only) Artifacts: 4 Relics: 0 Rubbing Rewards: 1(**) Tomb Raider Anniversary: walkthrough Tomb Raider Anniversary LEVEL 10: OBELISK OF KHAMOON From choosing coloring techniques, colour selection, and styling – your will love your new you.Īs for the maintenance, you should use a color-safe shampoo and conditioner, and protect your hair from the sun and protective styling products. So, no matter if you wish to have highlights or balayage, we are here to make your dream look come true. We love to enhance confidence and add memorable moments to any event of your life. At Canvas Hair & Nail Salon, we put your transformation needs first and foremost. Highlights can sometimes look harsh against balayage’s more natural looks. While highlights show the color at its most vibrant, balayage emphasizes the natural texture of the hair. Remember that both highlights and balayage require color treated hair, but there are some key differences between the two. In fact, it totally all comes down to what you want your hair to do, and how you want to style it. With different hair types, texture, and needs, what type of hair color treatment is right for your hair type depends entirely on your hair's natural color and the underlying health of your hair. Which one should you choose – Highlight or Balayage? As with most other hair coloring treatments, balayage and highlights require long-term care in order to maintain healthy, beautiful, color-treated hair. Understanding which type of hair color requires the highest level of maintenance can help you choose the right technique for your hair type. Highlights and balayage are both popular hair color techniques on the market today, but they both have their own set of pros and cons. So, be sure to spend some time researching for the best salon in Vancouver which provides you with the best consultation and balayage highlights service from an experienced hairstylist plus high quality products. Easiest to maintain since roots are less noticeable when hair grows out, you'll never have to worry about touch-ups anytime soon (some coloristas visit a salon every 3-6 months for retouching!).However, balayage typically starts on the hair away from the roots and works up towards the mid-shafts and ends of hair. Hair color can be played up or down as desired.One of the most popular methods of hair coloring.Are’t as natural looking, compared to balayage.All breaching processes can leave your hair dry and damaged, but, with dedicated hair products during coloring and after care, you’re able to get smooth, shining, and healthy-look highlights. Do highlights damage your hair? – Yes. Perfect for when you want a change, but don't want to commit.Add color/s to your hair without having to dye the full head.Can be warm or cool to match your specific style preference.Provide your hair with a dramatic boost in dimension, even on the darkest of hair colors. Pros and Cons of Highlights Highlights pros: You can get regular highlights, but balayage is a type of highlight that is most popular with creative glamorous colourists who do highlights that vary in depth and colour intensity. Your hairstylist creates a "bone structure" of colours in the hair and applies them to your hair strand by strand, alternating in order to give your hair a softer, more gradual transition. This technique will add layers, leaving a sun-kissed look.īalayage is a specific type of highlights, actually. For example, if you had blonde highlights, you would have several small highlights evenly spaced all over your hair. Highlights are created by lightening your hair to a level that is lighter than the base color. What's the difference between highlights vs balayage ? Which technique is the right fit? How do you maintain color-treated hair? Find out everything you need to know about these two popular hair coloring techniques here! Special edition: Pro tips from our stylist at Canvas Hair & Nail Salon – the best salon in Vancouver. Highlights and balayage are two of the most popular hair coloring techniques on the market today, but they each have their own set of pros and cons. When faced with adversity, the arts always find a way. Show was directed by a new member of the department - Kitty Keim.Ĭollege of the Siskiyous Fall 2020 Production – Zooming Into a New Age With Shakespeare Show was performed via Zoom and the playright even joined us for more than one showing. Roller skating Nanas, kids with tin cans, literary obsessed vampires, clumsy security guards, kids without tin cans, quirky game show hosts, and more. "Night Night, Roger Roger" by Megan Roni Ragone is a comedic fanciful play about the world after the sun goes down. In April of 2021 the College of the Siskiyous Theater department was the second theatre company to do a production of "Night Night, Roger Roger" by Megan Roni Ragone. In April of 2022 the Theatre Department presented "Almost, Maine" directed by Kathryn Young.Ĭollege of the Siskiyous Fall 2021 Production - Edward IIĬOS Theatre Department started off the school year 2021 with "Edward II," directed by Kitty Keim and written by Christopher Marlowe (one of Shakespeare's contemporaries), this show depicts an amoral and ruthless political world, in which each player single-mindedly pursues their own pleasures and ambitions, with dire consequences.Ĭollege of the Siskiyous Spring 2021 Production – "Night Night, Roger Roger" With a talented cast and crew, some amazing sets and some fun with puppets, this was a fun show for the whole department.Ĭollege of the Siskiyous Spring 2022 Production - Almost, Maine In the fall of 2022 the Performing Arts Department at College of the Siskiyous presented Tom Stoppard's comedic tour-de-force "Rosencrantz and Guildenstern Are Dead." This is a fabulously inventive tale of Hamlet as told from the worm's-eye view of the bewildered Rosencrantz and Guildenstern, two minor characters in Shakespeare's play. We were thrilled to have current students, former students, high school students, and community members join our cast and crew for a production that was truly magical.Ĭlose to 900 local elementary and middle school student were treated to special matinee performances and they seemed to love it!Ĭollege of the Siskiyous Fall 2022 Production - Rosencrantz and Guildenstern are Dead Audiences were transported to an island far away with pirates and mermaids and one hungry huge croc. In April of 2023 Performing Arts brought this back-story of Peter Pan to life, directed by Kathryn Young and Neil Carpentier-Alting. College of the Siskiyous Spring 2023 Production - Peter and the Starcatcher Fixed a bug where Software Update… would continue offering updates in the same session in which you had held down the Option key.Fixed a regression where the Entourage scripts could not be installed.Fixed a regression where SpamSieve wasn’t able to reset the corpus.Fixed a regression that could cause an error when switching applications.Added defensive coding to try to prevent a crash when saving the corpus to disk.Worked around a bug in Mac OS X 10.6 and 10.7 that could cause a crash.Worked around a bug in Mac OS X 10.11 that could cause a crash during Software Update….Previously, the process (and your fan) would keep running forever and possibly freeze the mail client. If a PDF file attached to an e-mail triggers an OS bug that leads to an infinite loop, SpamSieve now aborts the spamsieve-reader process after 30 seconds.Added "(?)" to error text to help people know what "Help button" refers to.Improved the error message when your version of SpamSieve is too old for the current version of Mac OS X.When SpamSieve encounters a permissions error, it now reports on any applicable ACLs (Access Control Lists).Why does a message disappear or go back to the spam mailbox after I train it as good?.What can I do if Apple Mail is crashing?.Setting Up Gmail, iCloud, Yahoo, AOL, or Other Web Mail.Automatically Deleting Old Spam Messages.Improved the following sections of the manual:.Should I download SpamSieve from this free site?.Added the following sections to the manual:.The About, Software Update, and help windows now use the San Francisco font on Mac OS X 10.11.Modernized code for the Xcode 7 compiler and the Mac OS X 10.11 SDK.Added support for App Transport Security on Mac OS X 10.11.Added the Outlook - Filter Mailboxes script as a workaround for automatically filtering messages with Outlook 2016.Please see this forum post for more information and workarounds for using SpamSieve with Outlook 2016. We recommend using the "send-a-smile" button in Outlook’s main window (below the search field) to send Microsoft feedback, asking them to prioritize this feature. This makes it impossible to create a rule that automatically processes incoming messages with SpamSieve. Microsoft Outlook 2016 removes the Run AppleScript rule action feature. Made various changes to improve SpamSieve’s filtering accuracy.Otherwise, with the SpamSieve plug-in not installed, Mail will move good messages to the spam mailbox without consulting SpamSieve. In any case, if you don’t see the SpamSieve commands in Apple Mail’s Message menu, be sure to choose Install Apple Mail Plug-In from the SpamSieve menu. It’s easiest if you update to SpamSieve 2.9.21 before updating to El Capitan. Works with Apple Mail under Mac OS X 10.11 El Capitan. SpamSieve supports AppleScript, so you can connect it to additional apps and fully integrate it into a custom workflow.SpamSieve only marks spam in your email client so you never lose any mail. Unlike the brute-force filters on servers, SpamSieve learns from your. SpamSieve understands what your spam looks like in order to block it all, but also learns what your legitimate messages look like to avoid confusion. C-Command Software’s 30 SpamSieve has been around since 2002 and is hands down the best spam filtering software for Mac. Turn off a new-mail notification in your email program, and let SpamSieve notify you only when you receive non-spam messages. SpamSieve for Mac SpamSieve is a robust spam filter for major email clients that uses powerful Bayesian spam filtering.SpamSieve can decode and look inside these messages.Customizable the allowlist and blocklist.Automatically maintains an allowlist to guarantee that messages from particular senders or mailing lists are never marked as spam without cluttering your address book with these addresses.Automatically maintains a blocklist to instantly adapt to spam messages from particular addresses and catch 100% of them.
The knight had a new ink blob effect to them. idk how i was just pressing e and arrow keys to see my new attacks since (SPOILERS) Great game! but i was playing as the knight in the area right after the deaf monster and i managed to break the game. The story is interesting and I think only works the way it's written now and told in the narrator-style the game does it in, so keeping the game as is but changing the plot/the way it's told wouldn't work. That being said, to reiterate: the voice acting, sound, visuals, and story (isolated from game length) are very well done. The problem is that while the voice acting is excellent, the sound and visuals give off an amazing vibe, and the plot is cool, the gameplay just isn't engaging enough for me to want to sit down for 3-5 hours. That's already not how I like to play games tho, so it's not completely fair of me, but still. If the story was kept as is but the game made shorter I think I would've liked it more. Now on my second session, and I can definitely see how this game is meant to be played in 1 sitting: it's very hard to keep track of and absorb the story if you don't. Even so, don't worry about trying it out, it has great qualities.Įdit: having now read a summary of the story, I can say it's powerful and very emotional. For me, even if it's a relatively short game, the balance between gameplay and plot is off since the gameplay isn't interesting enough to fill the gaps between the narrator bits. TLDR: great game, but your mileage may very depending on how you play games and if the gameplay is enough to hook you. Features CORSAIR iCUE RGB dynamic lighting functionality for PC.Playable in 14 languages, including English, French, German, Russian, Spanish, Italian, Arabic, Dutch, Japanese, Korean, Brazilian Portuguese, Turkish, Simplified and Traditional Chinese.Featuring the vocal talents of Andrew Dennis and Cassandra Lee Morris.Explore every nook and cranny to find secret passages and collectibles.Discover a 3-5 hour emotional story, intended to be played in a single-sitting on a dark, stormy night.Lure enemies into lethal traps, solve environmental puzzles and utilise your scythe, hookshot and pickaxe to avoid a gruesome end!.A child uncovers the past of an eerie house. A knight adventures into the depths of a deteriorating castle.3 main characters, each with their own unique gameplay styles.Venture through a hauntingly atmospheric pixel art world.**INMOST is an intimate story of loss and hope that some may find upsetting. A wanderer searches for answers.INMOST, by Lithuania-based indie studio Hidden Layer Games, is an emotional and deeply atmospheric narrative-driven puzzle platformer.Įxplore a crumbling, nightmarish landscape, slice through enemies, and spring deadly traps in order to escape the evil that awaits… Uncover the story of an adventurous young girl, a stoic knight and a man in search of answers.Įxplore a crumbling, nightmarish landscape, slice through enemies, and spring deadly traps in order to escape the evil that awaits… INMOST, by Lithuania-based indie studio Hidden Layer Games, is an emotional and deeply atmospheric narrative-driven puzzle platformer. Om detta spel Something is lurking in the shadows. For example, if the Inputs are in the left-hand column and its corresponding outputs are in the right-hand column. The replacement must be in-place and do not use any extra memory. Implement next permutation, which rearranges numbers into the lexicographically next greater permutation of numbers. If such arrangement is not possible, this method will rearrange it as the lowest possible order (That is actually, sorted in ascending order). If you have to create a bad API to satisfy LeetCode or whatever, write the good API first, and then wrap it. Step-5 reverse the array from the pivot+1.Suppose we want to implement the next permutation method, that method rearranges numbers into the lexicographically next greater permutation of numbers. nextpermutation is perfectly fine as a free function. Step-4 swap the pivot number with its exact largest one.Īfter the swap, the array looks like this num vector permobjects // fill vector do.The replacement must be in place and use only constant extra memory. Implement next permutation, which rearranges numbers into the lexicographically next greater permutation of numbers. Since the string you start with ( 'xxxxxoooo') is actually the last permutation of that string's characters in lexicographic order, your loop stops immediately. If such an arrangement is not possible, it must rearrange it as the lowest possible order (i.e., sorted in ascending order). 1 Answer Sorted by: 18 std::nextpermutation returns the next permutation in lexicographic order, and returns false if the first permutation (in that order) is generated. The word 'permutation' also refers to the act or process of changing the linear order of an ordered set. You can use that overload and specify a custom camparator that will sort by only the index. Description: Implement next permutation, which rearranges numbers into the lexicographically next greater permutation of numbers. In mathematics, a permutation of a set is, loosely speaking, an arrangement of its members into a sequence or linear order, or if the set is already ordered, a rearrangement of its elements. Step-3 if the first number pivot is found, find the exact largest number than the first number pivot from the right side.įrom the num array the exact largest number of pivot=1 is 2 std::nextpermutation has an overload that takes a comparison object and uses it to compare the elements of the range to get the next permutation. To add to dasblinkenlight, here is an example implementation. Ive done it for a programming competition many years ago, its only a couple dozen lines of code. Here, we will do this program by using the inbuilt nextpermutaion() function present in STL. It is not there, but the STL version is very easy to translate. This method takes a list as an input and returns an object list of tuples that contain all permutations in a list form. Step-2 if the pivot is not found, all numbers are in ascending order from the right side, which means the given permutation is the last permutation. Permutations enumerated through std::nextpermutation form a cyclic sequence without a beginning or an end, which means that you can call std::nextpermutation indefinitely and it will cycle through the same sequence of 120 permutations again, again and again. For example, lexicographically next permutation of acb is bac. Implement the next permutation, which rearranges the list of numbers into Lexicographically next greater permutation of list of numbers. First import itertools package to implement the permutations method in python. Step-1 find the first number pivot which not increasing in ascending order, from the right side.įrom the num array, 1 is the number that is not increasing in ascending order Lets understand the algo above one in a clear way with an example. Find the largest index k such that nums k such that nums The following algorithm is presented by a man named Narayan Pandita in the 14th century. No need to revert and use prevpermutation, and certainly no need to sort. we have to rearrange the array in the form of the next greater permutation in lexicographically or dictionary order. nextpermutation will step through all permutations, not only through greater permutations. from the first iterable until it is exhausted, then proceeds to the next iterable, until all of the iterables are exhausted. In this problem, we have given an int array. Today we will understand the 3rd problem from the SDE-Sheet which is the Next Permutation. Unfortunately, the stripping process is time consuming and often removes variable amounts of protein and antibodies to potentially give low quality, variable and, in some cases, artefactual data 20. This often requires regeneration of the membrane by stripping off the antibodies and reprobing 19. However, chemiluminescence is not amenable to multi-target detection when using antibodies with low specificity or for proteins that are very close in molecular weight with overlapping signals. Multiplexed target detection from chemiluminescent western blots is feasible when working with highly specific antibodies against targets that differ in molecular weight by at least five to ten kilodaltons (kDa). With limiting sample availability to assess components of microdissected tissue, sorted cell subpopulations, exosomes and primary cells cultured from small tissue specimens, the requirement to maximize quantity while preserving the quality of data is paramount 17, 18. However, a complete and concise guide to the workflow highlighting the importance of reagent selection, data workup and analysis remains elusive. These highly viewed and well cited articles support the need for updated guidance in producing quantitative western blot data. Recent articles have dissected and contrasted the old practices passed down from the early days of western blotting to offer revised methodologies that take full advantage of the currently available state-of-the-art instruments and reagents to achieve accurate, precise and reproducible data 14, 15, 16. The associated retractions from high profile articles 10, 11, 12, 13 support the unfounded reputation of western blotting as a difficult, frustrating and non-reproducible technique that is semi-quantitative at best. However, the original qualitative techniques and assumptions that have been passed down through generations of lab members were not formally updated to permit the production of semi-quantitative western blot data thus giving poor reproducibility and misleading interpretations 7, 8, 9. This permitted the generation of semi-quantitative data from chemiluminescence. During this time, the first camera-based imaging systems were introduced which enabled automated detection over a much broader linear dynamic range of about three to four orders of magnitude 6. Since the linear quantitative range of film is only about an order of magnitude and very challenging to assess saturation by eye, the results even between technical replicates were difficult to reproduce 5. Over the following two decades, as reagent and antibody technologies evolved, scientists attempted to produce quantitative results initially by scintillation counting and film densitometry 3, 4. Western blotting was first introduced in 1979 as a means of detecting a target protein in a sample with antibodies and detection using film 1, 2. The data reveal when and how to apply these detection methods to achieve reproducible data with a stepwise approach to data processing for quantitative analysis. A methodology to optimize and produce excellent, quantitative western blot results with rigorous data analysis from membranes probed with both fluorescent and chemiluminescent antibodies is described. Fluorescence reagent and detection solutions are the latest innovation in western blotting but there remains questions and debate concerning their relative sensitivity and dynamic range versus chemiluminescence. The development of sensitive, camera-based detection technologies coupled with an updated technical approach permits the production of reproducible, quantitative data. Chemiluminescence coupled with film-based detection was traditionally the gold standard for western blotting but accurate and reproducible quantification has been a major challenge from this methodology. Western blotting experiments were initially performed to detect a target protein in a complex biological sample and more recently, to measure relative protein abundance. |
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