General western blot protocol sample lysis preparation of lysate from cell culture 1. For western blots, incubate membrane with diluted primary antibody in 5% wv bsa, 1x tbs, 0. The phosphocreb s3 assay uses 2 labeled antibodies. Store at 20c phospho erk12 pathway sampler kit n 1 kit 5 x 20 l description. We used secondary antibodies with nearinfrared fluorescence detection odyssey ir imaging system, licor biosciences, in preference to chemiluminescence and ccd imaging, because the former shows substantially improved linearity and. I am trying to probe for phosphorylated erkthrough western blot. The kit includes enough antibody to perform two western blot experiments with each primary antibody. Protocols western blot complete western blot protocol phosphosolutions ultimate guide to doing western blots is broken down into 5 sections, containing a detailed list of steps, buffers, and specific materials needed within each section. Western blot analysis of a431 untreated and egf treated whole cell lysates probed with a mouse antiegf r antibody or b mouse antiegf r ptyr1148 antibody followed by detection with hrp conjugated goat antimouse igg 110,000, star207p.
Directblot hrp primary antibodies for western blotting. This western blot protocol provides a general procedure for use with the majority of biorad reagents. Best phosphoerk antibody sdspage and western blotting. The blot is a membrane, almost always of nitrocellulose or pvdf polyvinylidene fluoride. Place the cell culture dish on ice and wash the cells with icecold pbs. The phospho erk thr202tyr204 assay measures erk when phosphorylated at thr202tyr204. This antibody reacts with human, mouse, rat samples. In some cases specific recommendations are provided on product datasheets, and these methods should always be used in conjunction with product and batch specific information provided with each vial. Phosphoerk assays assay guidance manual ncbi bookshelf. Phosphorylated protein detection is more efficient by fluorescent western blot. Tested in western blot wb, immunofluorescence if, immunocytochemistry icc and immunohistochemistry ihc applications. Allow to polymerize before adding stacking gel by overlaying gently with etoh or nbutanol.
Ive used others before but they could take at least 56 hours to complete and i had to buy two kits one for total and one for phosphoerk, its an advantage if you have few samples. This protocol utilizes the 4g10 antiphosphotyrosine monoclonal antibody mab be. We used secondary antibodies with nearinfrared fluorescence detection odyssey ir imaging system, licor biosciences, in preference to chemiluminescence and ccd imaging, because the former shows substantially improved linearity and increased. Western blot analysis of wholecell extracts from unstarved wildtype mouse embryonic. Make 8 m stock of guanidinehcl mix 50% glycerol 25ml 5m nacl 2.
Please refer to primary antibody datasheet or product webpage for recommended antibody dilution a. Extracellular signalrelated kinase erk12 or p4244 is a kinase in the mitogenactivated protein kinase mapk family and phosphorylation of erk perk can be used as a common end point measurement for the activation of many classes of g protein coupled receptors gpcr. The phosphoerk thr202tyr204 assay measures erk when phosphorylated at thr202tyr204. The optimal dilution should be determined by titration for each individual assay of interest. Specificity and sensitivity of phosphop4442 mapk erk12 thr202tyr204 antibody. The erk12 signaling pathway is involved in the stimulation of.
As soon as lysis occurs, proteolysis, dephosphorylation and denaturation begin. Anti phosphoerk 1,2 db 0 western blot analysis of erk 1,2 activation in untreated pc12 cells a, cells treated with egf100 ngml, 5min b, pma100nm, 30min c in serum free dmem, and 8. Phospho specific monoclonal antibodies against each erk phospho form were obtained from sigmaaldrich figure 1c. When doing your western blot, you should use an alternative blocking agent, such as bovine serum albumin, or a proteinfree blocking agent such as advanblockpf. Methods in molecular biology methods and protocols, vol 661. Detecting phosphorylated erk12 sdspage and western blotting.
Western blotting of phospho proteins protocol homogenize the cells or tissue of interest in lysis buffer made fresh and containing a cocktail of protease inhibitors and phosphatase inhibitors when dealing with phosphorylated proteins. Human phospho kinase antibody array ordering information introduction analyzing the phosphorylation profiles of kinases and their protein substrates is essential for understanding how cells recognize and respond to changes in their environment. Western blotting principles and methods sigmaaldrich. General protocol for western blotting bulletin 6376 ver c useg 170657 0517 sig 1216 web site biorad. The western blot technique is a powerful tool to elucidate the complex signaling events that underlie biological processes and. By using htrf advanced phosphoerk thr202tyr204 only 300 cells are sufficient for minimal signal detection, while 10,000 cells are needed for a western blot signal. Western blotting is an analytical technique used to detect specific proteins in the given sample. The phospho erk12 pathway sampler kit provides an economical means of evaluating multiple members of the erk pathway as well as their activation state. This paper highlights critical steps in the western blot protocol and demonstrates how protocol changes can affect the final outcome of your blot. I want to ask you what is the best antiphospho erk12 antibody for western blot. Where appropiate, we refer to these handbooks in the text and provide links to the pdf versions. Fluorescent western blot protocol with azurespectra reagents. This chapter addresses the use of perkinelmers alphascreen surefiretmassay to measure erk phosphorylation.
Development of the strategy and protocol grew out of our study. In the figure below, phosphop4442 mapk erk12 thr202tyr204 antibodies are. Antiphosphoerk antibodies and in vitro phosphorylation. Regarding the methods for stripping membranes to reprobe for total erk subsequent to phospho erk, i recommend the mild stripping protocol from abcam. Storage and stability store at 28 c for up to one month.
Adavanced phosphoerk thr202tyr204 cellular htrf kit cisbio. May 01, 2012 extracellular signalrelated kinase erk12 or p4244 is a kinase in the mitogenactivated protein kinase mapk family and phosphorylation of erk perk can be used as a common end point measurement for the activation of many classes of g protein coupled receptors gpcr. Thermo fisher antibodies are validated for applications including western blotting, immunocytochemistry, flow cytometry, and chromatin immunoprecipitation. The phospho creb s3 assay uses 2 labeled antibodies. Pdf detecting tyrosinephosphorylated proteins by western blot. Effects of pma on perk12 determined by western blot. Western blotting detection of erk 12 in a twofold dilution series of pc3u cell lysate. The phosphocreb s3 assay measures creb when phosphorylated at s3. Phospho erk12 thr202tyr204 and erk12 antibodies were used in both detection assays. How different is western blot protocol for phosphorylated protein. Phosphorylation is a key response to cellular signaling and the phosphorylation status of a protein changes dynamically. Activation of erk extracellular signal regulated kinase requires the phosphorylation of thr202 and tyr204 amino acid residues.
Jurkat cells were treated with tpa, a phorbol ester that activates pkc to induce erk phosphorylation. The phospho erk thr202tyr204 assay uses 2 labeled antibodies. Regarding the methods for stripping membranes to reprobe for total erk subsequent to phosphoerk, i recommend the mild stripping protocol from abcam. Western blot detection of phosphorylation events best. Such modified proteinspecific antibodies can be used in a wide range of in vivo. Human phosphokinase antibody array ordering information introduction analyzing the phosphorylation profiles of kinases and their protein substrates is essential for understanding how cells recognize and respond to changes in their environment. Erk1 and erk2 are very similar in sequence around erk1s phospho site thr202. Set up your gel apparatus, prepare separating gel monomer. The phosphoerk thr202tyr204 assay uses 2 labeled antibodies. Like i heed to all the steps of my protocol, i think i have a problem with the antibody, and that is why i would like to know what is the best antibody for detect.
Phospho erk antibody, antiphosphoerk1 pthr202ptyr204 and erk2 pthr185ptyr187 antibody produced in rabbit. Phosphoerk12 thr202tyr204 and erk12 antibodies were used in both detection assays. The htrf advanced phosphoerk assay is at least 30fold more sensitive than the western blot, and shows optimal correlation. Homogenize the cells or tissue of interest in lysis buffer made fresh and containing a cocktail of. Phosphospecific antibodies affinity purified polyclonal antibodies total monoclonal antibodies total labeled antibodies total akt1 s473 af1775, af17751 mab1775, mab17752 akt2 s474 af2315, af23151 mab2315 akt3 s472 mab1463.
Western blotting antibodies for analytes related to the human. If not, p44a and p42 are probably together in same band. Physical form the product is provided as ascites fluid containing 15 mm sodium azide as a preservative. Phospho erk antibody, anti phospho erk1 pthr202ptyr204 and erk2 pthr185ptyr187 antibody produced in rabbit. Western blot analysis of extracts from 100 ngml hegf treated a431 cells. Western blotting could detect target protein which is as low as 1ng due to the high resolution of the gel electrophoresis and strong specificity and high sensitivity of the immunoassay. Updatedsummer2016jp page1 important considerations for kinase sample preparation success successful preparation of tissue or cells for the subsequent assay of kinase activity depends largely on the three. Further, the simple and rapid protocols of msd assays. Comparative analysis of erk phosphorylation suggests a mixed.
In wb, proteins are first separated based on size molecular weight via gel electrophoresis. Directblot hrp antierk12 phospho thr202tyr204 antibody. Phosphospecific monoclonal antibodies against each erk phosphoform were obtained from sigmaaldrich figure 1c. For western blots, incubate the membrane with antibody diluted in blocking buffer. If you are looking at both phospho and total protein on the same blot, look at the phospho form first before stripping. The antibody reacts specifically with as little as 0. The totalerk assay quantifies the expression level of erk in a cell lysate. Generation and purification of highly specific antibodies for detecting. Contrary to western blot, the assay is entirely platebased and does not require gels, electrophoresis or transfer. Mar 12, 2010 to be sure about p44p42 phosphorylated or not, treat blot membrane with a antierk12 antibody.
Theres a lot of erk elisa kit out there, but with this one you can record both total and phosphoerk levels in less than two hours. Here, we will highlight the critical steps in the western blot protocol and. Methods lysate preparation lysates of hek 293 and hela cells were prepared according to two techniques. How can i get phosphoprotein signal in western blot.
The total erk assay quantifies the expression level of erk in a cell lysate. The bioplex cell lysis kit was used to prepare cell lysates for western blot and bioplex analysis refer to bulletin 3033. Phospho proteins do not respond well after a stripping procedure. Anti phospho erk antibodies and in vitro phosphorylation. Western blotting a beginners guide western blotting identifies with specific antibodies proteins that have been separated from one another according to their size by gel electrophoresis. The phospho creb s3 assay measures creb when phosphorylated at s3. Western blotting wb is the most widely performed immunoassay and is the best initial validation technique used to identify proteins of interest within a tissue homogenate or cell extract. Measurement of map kinase activation by flow cytometry using.