Western blot protocol immunoblotting protocol sigmaaldrich. The membrane was probed with a c myc epitope tag polyclonal antibody cat. For western blot detection of myc tagged proteins, chromotek recommends. Immunoprecipitation of c myc protein from hela nuclear extracts using 5. Proteins were transferred to a pvdf membrane and blocked at 4c overnight. Western blot protocol ihc protocol if protocol facs protocol. Easyblot antimouse igg kit gtx22166701 was used in western blot. Cell lysate and conditioned media cells from 10 cm plates are are collected at 100% confluence, day 0, in 1 ml of western lysis buffer wlb and stored at 20.
The myc tag monoclonal antibody 9e10 displays highly. Pdf western blot protocol technical method researchgate. Chromotek myc tag monoclonal antibody 9e1 rat monoclonal 9e1 to myc tag. The basic coip protocol is the same as that described for ip, and indeed any. Clone 9e10 shows sequence context bias toward 6xmyc tagged proteins.
Specificity the antibody recognizes the myc tag sequence eqkliseedl at the nterminus, cterminus, or internal site of the fusion protein. The protocol includes only three steps and is quick and easy to perform. How to plan an ip of your myctagged protein when using the chromotek myc trap. To perform the assay, sample is incubated with a capture antibody conjugated with a proprietary tag and a second detection antibody linked to hrp, forming a sandwich with c myc in solution. Perform electrophoresis according to standard protocols 3. Antimyc and antiddk monoclonal antibodies ta150014.
Sensitivity of rat c myc antibody 9e1 goes down to 10 ng of purified gfp myc and myc gfp. If the signal is strong, expose the film for 30 seconds or less. Sep 01, 2017 unfortunately, however, the purified 9e10 scab did not bind to a protein with a myc tag in a western blot format. Please refer to the western blot protocol found on the product web. Southern and northern blot, dot blot, dna fingerprinting. A myc tag can be used in many different assays that require recognition by an antibody. The antibody recognizes the myc tag fused to either the amino or carboxy terminus of targeted proteins in transfected cells. Optimization each western blot system must be optimized to obtain consistently good results. Detection and purification of backbonecyclized proteins. Western blot analysis of 12 tag protein lysate, using myc tag antibodycat. The antibody detects transfected proteins contanining c myc tag. Hela transfected lysates were subjected to immunoprecipitation using b normal mouse igg or c 2. Chemiluminescence western blotting technical guide and protocols. The guide is intended to be an educational resource to introduce the method rather than a benchtop protocol, but a more concise document.
The precipitated protein was detected by gtx628259 diluted at 1. Myctag 71d10 rabbit mab detects recombinant proteins containing the myc epitope tag. Application antic myc tag antibody produced in rabbit has been used in western blotting. How to plan an ip of your myc tagged protein when using the chromotek myctrap. This antibody reacts with recombinant protein samples. Choosing the appropriate methods and reagents is important for obtaining a good result. Western blot analysis of extracts from hela cells 48 hours following mock transfection, transfection with nonspecific control sirna or transfection with c myc sirna. Western blot analysis of extracts from cos cells, untransfected or transfected with myc chorionic somatomammotropin hormonelike 1 csl, using myc tag 9b11 mouse mab hrp conjugate. Relevance epitope tags are short peptide sequences of known.
This product has been approved for use in this application by cst. General western blot protocol sample lysis preparation of lysate from cell culture 1. These tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, although they also find use in antibody purification. Singlestep detection of c myc tagged recombinant proteins by west ern blot. Clone 9e10 antic myc can then be used to detect the protein on western blot or to immunoprecipitate the recombinant protein when doing binding studies with transfected cells. Performed by loading 25 g of 293t cells transfected with c myc mycbp lane 1, transfected with c myc fli1 lane 2 and transfected with 12tag lane 3 onto an sds polyacrylamide gel. Western blot analyses showed that based on the results of the substitutional analysis it is possible to shorten and mutate the original myc tag without loss of sensitivity as determined by a dilution series with the scfv variants of the 9e10 antibody figure 2. Test for expression of your recombinant gene by western blot analysis or functional assay.
Antimyctag single domain antibody fragment vhh, nanobody. Myctag antibody rabbit polyclonal from proteintech validated in western blot wb, immunoprecipitation ip, immunofluorescence if, coimmunoprecipitation ip,enzymelinked immunosorbent assay elisa applications. Chemiluminescence western blotting technical guide and. Background epitope tags are useful for the labeling and detection of proteins using immunoblotting, immunoprecipitation, and immunostaining techniques. Biotin anticmyc epitope tag antibody anticmyc 9e10. Western blot analysis of cell extracts alone lane 1, cells expressing myc tagged to the carboxy terminus of the protein lane 2 and myc tagged to the amino terminus of the protein lane 3, using myctag antibody. Western blotting immunoblotting introduction many western blot procedures can be used for detection of recombinant fusion proteins with the appropriate apconjugated antibody. The guide is intended to be an educational resource to introduce the method rather than a benchtop protocol. A ten amino acid tag corresponding to amino acids 410419 of the human c myc sequence is frequently incorporated into recombinant proteins for a variety of purposes. Myc tag antibody western blotting analysis cmyc protein. Jan 28, 2020 we tested by western blotting six antibodies that recognize the c myc epitope tag, including monoclonal antibodies 9e10, 4a6, 9b11, and 71d10 and polyclonal antibodies 9106 and a14.
If there is no antibody against the studied protein, adding a myctag allows one to follow the protein with an antibody against the myc epitope. These are usually derived from viral genes, which explain their high immunoreactivity. General protocol for western blotting bulletin 6376 ver c useg 170657 0517 sig 1216 web site usa 1 800 424 6723 australia 61 2 9914 2800 austria 43 1 877 89 01 177 belgium 32 03 710 53 00 brazil 55 11 3065 7550. For western blot detection of myctagged proteins, chromotek recommends. Aug 07, 2012 western blot anti myc tag antibody chip grade ab92 all lanes. All displayed contextdependent differences in their ability to detect n or cterminal myc tagged proteins. The antibody recognizes the myc tag sequence eqkliseedl at the nterminus, cterminus, or internal site of the fusion protein. Singlestep detection of cmyctagged recombinant proteins by west ern blot. Western blotting overview 23 western blotting 10 steps overview 45 step 1 gel electrophoresis protein gel accessories 6 protein ladders 712 step 2 electrotransfer western transfer system 1112 transfer buffers and membranes protein stain kits 1415 signal enhancement 1517 step 3 blocking introduction 1819. Anti myc tag single domain antibody fragment vhh, nanobody. This c myc antibody clone 9e10 is useful for flow cytometry pmid. Immunoprecipitation ip technical guide and protocols.
When performing a western blot, its a wise idea to follow your procedure step by step. Western blot protocol with workflow steps for different blot procedures, describing the electrophoretic transfer of proteins from sds polyacrylamide gels to sheets of nitrocellulose. If you do not have an antibody to your protein, you may use the anti myc antibody or the antihis cterm antibody to detect your recombinant protein see page 11 for ordering. The following guide discusses the entire process of producing a western blot. The antibody may crossreact with c myc protein by flow cytometry and. As with other blotting applications, the far western method is effective for both membrane and ingel detection systems. B western blot with an anti myc antibody did not detect any signals in a total protein extract of cells transfected with k500mycgfp, while a band at 34 kda black arrow, which is likely to. Epitope tags include alfa tag, v5 tag, myc tag, ha tag, spot tag, t7 tag and ne tag. Western blotting protocol immunoblotting protocol western blotting refers to the electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to sheets of pvdf or nitrocellullose membrane, followed by immunodetection of proteins using antibodies with fluorescent or chemiluminescent detection. General protocol for western blotting protein separation by gel electrophoresis 1. Specificthe immobilized antic myc monoclonal antibody binds the c myc epitope tag with high specificity, providing high yield immunoprecipitation products and clean western blot detection high capacity excellent results with as little as 1 g of antic myc antibody in ip mode with the positive control lysate. Detection there are numerous ways to perform each step. F ubiquitylation status of ago1 was determined by western blot analysis of ip fractions using an antibody specifically raised against k63ub. Western blot procedures step by step sino biological.
Western blotting handbook thermo fisher scientific. If there is no banding, expose the film for 5 minutes, then 30 minutes and up to overnight if the signal is weak. Anticmyc tag antibody myc tag antibody sigmaaldrich. Use in immunoblotting reported in scientific literature pmid. To perform a western blot successfully, every single step should not. Mouse monoclonal antibody clone 9e10 conjugated to peroxidase. Western blotting protein blotting or immunoblotting, is an important technique to detect specific proteins in a sample of tissue homogenate or extract. The antibody recognizes the c myc tag sequence at either the n or the cterminus of the fusion protein. How to plan an immunoprecipitation of your myctagged. Dissect the tissue of interest with clean tools, on ice preferably, and as quickly as possible to prevent degradation by proteases.
Western blot protocol immunoblotting protocol sigma. Fulllength western blotting protocol 83 using chemiluminescent substrates recommended reading 84. Click here for onbead digest protocol for mass spectrometry. Western blotting procedure the procedure for western blotting is as follows. The myc tag monoclonal antibody 9e10 displays highly variable.
The lysate contains the commonly used epitope tags ha, c myc, gluglu, gst, 6his, hsv, t7, v5, vsv, e tag, s tag, and flag tag. Posi tag is a recombinant protein that contains 12 different tags. Blot stripping protocols blot storage blot storage protocol troubleshooting no signal or weak signal high uniform background nonspecific bandswrong size or multiple bands speckled or swirled background other issues benchtop western blotting protocol reference recipes western blot reagents available from novus biolgoicals 12 36 7 78 910. Perform electrophoresis according to standard protoc. The following general protocol is included for your convenience. To compare the expression levels of different globin proteins, c. Place the tissue in roundbottom microcentrifuge tubes or eppendorf tubes and immerse in liquid nitrogen to snap freeze. Here, we will highlight the critical steps in the western blot protocol and demonstrate. The antibody recognizes the myctag sequence eqkliseedl at the nterminus, cterminus, or internal site of the fusion protein. The fastscan total c myc elisa kit is a sandwich enzymelinked immunosorbent assay elisa that detects endogenous levels of c myc. Recombinant protein with histag, gsttag, myc and dykddddk anchor. Epitope tag and loading control antibodies 2627 step 5 wash washing the membrane 28. Easyblot antimouse igg gtx22166701 was used as a secondary reagent. It has a predicted weight of 45 kda and is intended for use as a positive control in western blot applications.
Examples are cellulite localization studies by immunofluorescence or detection by western blotting. To purify your recombinant protein, you may use metalchelating resin such. Western blot protocol ihc protocol if protocol facs protocol immunoprecipitation ip protocol ip protocol with magnetic beads related products 15 ta150121s. Recombinant protein with his tag, gst tag, myc and dykddddk anchor. Other protocols and detection reagents are suitable. How our antibodies and protocols help you get accurate results. Antimyc and antiddk monoclonal antibodies ta150014 origene. Place the cell culture dish on ice and wash the cells with icecold pbs. Western blot procedures step by step western blotting protein blotting or immunoblotting, is an important technique to detect specific proteins in a sample of tissue homogenate or extract. For immunoprecipitation of protein interactions involving cmycta.
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