No, you can't. Elisa TMB after enzymatic coversion is soluble. Your blue colored product will be washed away. You have to use special substrate fo Western Blot that precipitates or emits chemiluminescence after coversion with HRP.
Best Regards
No, you can't. Elisa TMB after enzymatic coversion is soluble. Your blue colored product will be washed away. You have to use special substrate fo Western Blot that precipitates or emits chemiluminescence after coversion with HRP.
Best Regards
Renu:
That is not possible, since TMB is a SOLUBLE reagent, and you need a chromogen that PRECIPITATES at the reaction band. You need a reagent like H2O2/4-Cl-a-naphthol.
Alternatively, you can use a chemoluminiscent system like H2O2/luminol, in order to improve sensitivity.
Good luck!
Actually, Sigma Chem Co sells a product called "TMB for membranes (T0565)" which leaves a blue colored deposit. I have not used this in Western blots but it seems to work well on PVDF membranes in an electrochemical format catalyzed by immunoperoxidase. Their chemical trick is not explained, but I suspect the blue-colored charge-transfer complex is being stabilized somehow.
Dear Renu,
Self made ECL will cost you 46.50 EURO for luminol, 13.80 EURO for p-Coumaric acid (Sigma). TRIS, DMSO, peroxide you will find everywhere. It will be enough for 5 (five!!!) liters (!!!) of Western -Blot developing solution it means by consuming 10ml (reasonable) per mini-gel/blot the cost of 0.1 Euro per blot...
Just follow the protocol below. My tips are - TRIS doesn't need to be very accurate (pH electrode canot measure it anyway...), keep luminol and coumaric acid frozen in DMSO, prepare all the solution fresh just before use.
Keep fingers cross - Jan
http://www.westlab.org/protocols/Homemade%20ECL.pdf
There is an excellent and very sensitive ready to use subtrate: http://www.seramun.com/products/substrates/membrane-substrates.html
No, you can't. TMB is soluble chromogen. You must use non soluble chromogens, for example 4-chlor-1-naphtol, o-dianizidine, 3,3-diaminobenzinine-tetrahydrochloride, 3-amino-9- ethyl-carbazol.
Hi Liubov,
sorry, you can use TMB as a soluble substrate for ELISA and as an insoluble substrate in blottings. There are companies offering both ... and the sensitivity is higher in western blots compared to DAB + Ni-ions (NiCl2) or Co-Ions. The background is excellent.
http://www.seramun.com/products/substrates/membrane-substrates.html
Oxidized TMB will precipitate either at high concentrations (let the reaction run much longer without stopping and you will see this as small long needles) or at disitinct pH and ionic conditions. I used this also in the development of many commercial assays. Try this from the website. It's excellent.
For making soluble TMB product of ELISA into insoluble one you have to add 0.05% sodium nitroprusside to ELISA TMB substrate. Thereafter you can use for western blot.
http://www.ihcworld.com/_protocols/chromogen_substrates/POD_TMB_blue.htm
You can precipitate oxidized TMB from the group consisting of alginic acid, methyl vinyl ether-maleic anhydride copolymer, dextran sulfate, carrageenan and combinations thereof, wherein the colored product forms a stable precipitate with the polymeric compound. I am using dextran and I found it from a TMB formulation for soluble and precipitable HRP-ELISA patent.
http://www.google.com/patents/US5006461
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