I am searching for a protocol for alkaline phosphotase staining for zebrafish embryos. Insufficient draining of the blot may alter substrate performance. Hsp70 bands should show up within a couple minutes. Problems with nbtbcip visualization sdspage and western. Bcipnbt alkaline phosphatase substrate solution blue. Nbt nitroblue tetrazolium dye used for western blot. Thermo scientific pierce nbt and bcip substrates are powders and readymade solutions of nitroblue tetrazolium and 5bromo4chloro3indolyphosphate for chromogenic blot and ihc staining with alkaline phosphatase probes. In a sidebyside comparison, western blot results demonstrate that alkaline phosphatase substrate bcip nbt from scripps laboratories works faster and identifies more bands than substrate from 3 leading biochemical suppliers. Western blotting analyses using alkaline phosphatase detection all steps should be done at room temperature with agitation.
Strip northern and southern blots thermo fisher scientific. For a more detailed protocol, please reference the. The combination of nbt nitroblue tetrazolium chloride and bcip 5bromo4. Add readytouse bcip nbt solution to cover well the blot. After blotting antigen conjugates to the antibodyalp in western blotting, the targeted protein is detected with high sensitivity. Whether using bcipnbt or dab, blocking the membrane with bsa or other materials is required to minimize nonspecific signals. Can anyone give a procedure for nbtbcip staining for zebrafish embryos. General western blot protocol sample lysis preparation of lysate from cell culture 1.
This solution volume is suitable for one 10 cm x 10 cm piece of membrane. Western blot colony and plaque lifts in situ hybridization. After blotting antigen conjugates to the antibodyalp in western blotting, the targeted protein is detected with high. Shelf reagent storage temperature life ap color development 20 c 1 year reagents, bcip and nbt desiccated tris buffer solution 2325 c 1 month use sodium azide as a bacteriostat bcip and nbt stock solution 4 c 3 months store in amber vial, protected from exposure. Download troubleshooting handbooks for ihc, western blot and elisa for free.
This bcipnbt 1 component buffered alkaline phosphatase substrate 5bromo4chloro3indolylphosphate and nitroblue tetrazolium is a precipitating substrate used with the enzyme alkaline phosphatase designed for various membrane immunoassays but not recommended for microwell or immunohistochemical applications. Bcipnbt color development instructions for preparing. Ecl western blotting substrate technical manual, tm317. The principle of the western blot is relatively simple proteins are first separated. Test for stripping efficiency for biotinylated probes detected using north2south chemiluminescent hybridization and detection kit product no. We use this protocol for the routine testing of our antibodies. For chromogenic detection of membranebound proteins. Alkaline phosphatase ap conjugated antibodies catalog nos.
Bcip nbt alkaline phosphatase substrate solution blue. Bcipnbt alkaline phosphatase substrate protocol nb90078646 find general support by application which include. The intermediate is then oxidized by nbt to produce a dimer, which is an intense insoluble purple dye. The substrate is a stabilized formulation of bcip 5bromo4chloro3indolyphosphateptoluidine and nbt nitroblue tetrazolium chloride. Biorad offers a complete line of reagents to meet virtually every possible. Bcip nbt membrane alkaline phosphatase substrate is an artificially manufactured chromogenic substrate for use in sensitive colorimetric assays. Bcip nbt system is based on hydrolysis of bcip and reduction of nbt producing a deep purple reaction product. Solutions for any blotting application choose the best approach for your needs when it comes to western blot detection, you can follow a number of different paths. Protocols, optimization tips, troubleshooting guides, and more for western blot.
How do you choose the right western blot detection method. Remove the 1x tbst buffer from the hybridized western blot and add 20ml ap buffer. Has anyone been using 1step nbtbcip substrate solution. Bcip nbt alkaline phosphatase substrate solution protocol. The protein sample to be examined is separated by sds page and transferred to a nitrocellulose membrane by semidry electro blotting using 0. The reaction product is extremely stable and does not fade when exposed to light.
Product specifications for each apconjugated antibody, western blot experiments with 100 ng of recombinant. Make sure you spin down the antibodies before diluting into buffer. The intense color can be observed visually, is very stable, and will not fade upon exposure to light. Bcipnbt alkaline phosphatase substrate solution protocol. But, this results in a higher background and magnesium salt precipitation also starts to build up. If you are unable to obtain the desired information please contact vector technical service or your local vector labs distributor for further assistance. Western blue stabilized substrate for alkaline phosphatase is a stable, readytouse substrate for western blots and immunoscreening e. Destaining stained protein bands for western blot analysis 1 2 3 21.
Buffer contains alp catalyst for the same detection sensitivity. Selected vector kit instructions and associated protocols are listed below in pdf format. Reagents, bcip and nbt desiccated tris buffer solution 2325 c 1 month use sodium azide as a bacteriostat bcip and nbt stock solution 4 c 3 months store in amber vial, protected from exposure to light instructions for preparing bcipnbt color development solution catalog numbers 1706532 and 1706539. I usually blot onto pvdf and detect the primary antibodies with alkaline phosphataseconjugated secondaries. Our group is looking at moving away from traditional western blot methods as there are many more time. Nitroblue tetrazolium nbt is used with the alkaline phosphatase substrate 5bromo 4chloro3indolyl phosphate bcip in western blotting and immunohistological staining procedures. Antibody conjugation each apconjugated antibody was prepared by crosslinking the appropriate primary antibody with alkaline phosphatase using glutaraldehyde harlow and lane, 1988. The developed membrane can then be imaged or stored for subsequent use.
View specific protocols for bcipnbt alkaline phosphatase substrate nb90078646. The colored product can either be of the nbt bcip reaction reveals where the antibody is bound, or can be used in immunofluorescence. In principle, one can continue color reaction in 4c overnight. Check out how our product, protein detector bcipnbt western blotting kit 25 blots can help with research, development, validation, and lab testing. Has anyone been using 1step nbtbcip substrate solution for developing western blots. It is recommended that if nbtbcip fails to produce desirable stain in 3. But i still have to get rid of the blue precipitate from the apreaction. Colorimetric alkaline phosphatase and peroxidase substrate detection systems. Detect proteins by adding alkaline phosphatase buffer 100. Bcipnbt substrate bcip nbt substrate is a readytouse single component substrate intended for the detection of alkaline phosphataselabeled probes on membranes e. The blot can be developed until the signal of the bands reach the intensity required, this allows for a good level of sensitivity as the development of the blot can be observed directly and the reaction halted immediately by washing off the substrate. Nbt serves as an oxidant and bcip as the alkaline phosphatase substrate. A good way to optimize this is to spot serial dilutions of your primary onto membranes, then block, detect, and wash with secondary.
It is recommended that if nbtbcip fails to produce desirable stain in 3 hours, one can simply use bm purple instead. Bcipnbt produces a purple reaction product which is soluble in alcohol. Detecting mirnas by liquid hybridization and color development. Bcipnbt 1 component membrane alkaline phosphatase substrate. Chromogenic western blotting substrates thermo fisher. Phosphatase the standard protocol for western blotting is as follows.
Bcipnbt system is based on hydrolysis of bcip and reduction of nbt producing a deep purple reaction product. Introduction many western blot procedures can be used for detection of recombinant fusion proteins. Seracare is a trusted partner and worldwide supplier to the diagnostic testing industry, advancing the development of clinical genomics, molecular, and serological diagnostics. For details, see the protocol described in the roche. The nbtbcip reagent is also commonly used in chromogenic western blot immunoassays. For color development, bcipnbt and dab are widely used chromogenic substrates for ihc, while ecl and sswf are commonly used chemiluminogenic substrates for western blot. Ideally though, chromogenic western blots with ap use a combination of nbt and bcip that result in an intense, blackpurple precipitate which provides a greater substrate sensitivity. Prepare dab or bcip nbt substrate solution described below. Protein detector bcipnbt western blotting kit 25 blots. 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. In a sidebyside comparison, western blot results demonstrate that alkaline phosphatase substrate bcipnbt from scripps laboratories works faster and identifies more bands than substrate from 3 leading biochemical suppliers. The nbt bcip reagent is also commonly used in chromogenic western blot immunoassays. There are several counterstains possible in combination with bm purple or nbt bcip in general, for instance, fastgreen fcf or nuclear fast red. The blot is a membrane, almost always of nitrocellulose or pvdf polyvinylidene fluoride.
Mar 23, 2008 make sure you spin down the antibodies before diluting into buffer. To 10ml of this add 33l bcip t 5bromo4chloro3indolyl phosphate, ptoluidine salt, make up 50mgml in water or dimethyl formamide. This western blot protocol provides a general procedure for use with the majority of biorad reagents. The ecl western blotting substrate detects picogram amounts of antigen, and with the use of photographic or other imaging methods, visualizes the presence. I have seen an impressive demonstration of using a few ml of 1step nbtbcip substrate solution to detect protein bands on the membrane. Tmb hrpo membrane substrate is a ready to use solution that needs no preparation or dilution. Bcipnbt membrane alkaline phosphatase substrate nbt100. Together nbt and bcip form reactants in the presence of alkaline phosphatase which yields a dark purple to black, waterinsoluble, precipitant product providing strong sensitivity.
The uptima bcipnbt solution substrate is a one component chromogenic. Western blot protocol antibody company, buy antibodies online. In the presence of nbt, an insoluble dark blue precipitate is formed after a reduction reaction instead. The commonly used enzyme labels are horseradish peroxidase and calf intestinal alkaline phosphatase, which results in catalyzing their respective substrates 3,3. Incubate tissues with alkaline phosphatase conjugated secondary antibody for 30 minutes at room temperature in a humidity. It is recommended that you allow the substrate solution to equilibrate to room temperature before use. Standard protocol for western blots with alkaline phosphatase staining. Take advantage of thermo scientific lab vision bcipnbt for western blot ready touse, specially formulated for use with alkaline phosphatase enzyme systems. Incubate with nbtbcip provided for 10 seconds to 2. Problems with nbtbcip visualization protocol online. The ecl western blotting substrate is a highly sensitive nonradioactive, enhanced luminolbased chemiluminescent substrate for the detection of horseradish peroxidase hrp on immunoblots. It has turned out to be very robust and suitable for almost all our antibodies. Mix the reagents just before use and store in the dark. Tmb hrp membrane substrate one component ready use.
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. The colored product can either be of the nbtbcip reaction reveals where the antibody is bound, or can be used in immunofluorescence. The solution is used for the sensitive detection of alkaline phosphatase in blotting protocols including. Place the cell culture dish on ice and wash the cells with icecold pbs. Bcipnbt membrane alkaline phosphatase substrate is an artificially manufactured chromogenic substrate for use in sensitive colorimetric assays. The ecl western blotting substrate is a highly sensitive non radioactive. Add readytouse bcipnbt solution to cover well the blot. The bcip and nbt stocks will last indefinitely when stored at 4c or 20c. Western blot protocol antibody company, buy antibodies. Pour 20 ml of solution a, 10 ml of solution b, and 30 ml of deionized water included in rapid cbb destain kit into a disposable tray.
Chromogenic detection for western blot, ihc, and elisa. Bcip is hydrolyzed by alkaline phosphatase to form a blue intermediate. Substrate buffer containing 80 l bcip solution and 60 l nbt solution per 10ml and is mixed freshly for each staining. Alkaline phosphatase chromogen bcipnbt ready to use abcam. Western blue stabilized substrate for alkaline phosphatase. Westernview detection kit basic protocol incubate with primary antibody, place the membrane in a plastic tray and wash. Can anyone give a procedure for nbtbcip staining for. Identical gels and nitrocellulose blots of il6 protein samples 0. Alkaline phosphatase hydrolyzes the bcip to 5bromo4chloro3indole, which is oxidized by oxygen in the atmosphere, yielding a blue dye. Nbtbcip substrate combinations with ap produce sharp bands with little background staining which is. I have already found a protocol for the stripping procedure itself biochem. Immunodetection using bcipnbt substrate sigmaaldrich. Alkaline phosphatase chromogen bcipnbt ready to use.
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