Tetramethylbenzidine as precipitating HRP substrate

David Micklem dmicklem at cmgm.nospam.invalid
Thu Dec 19 15:35:13 EST 2002

In article <3E018409.CC016B0 at neuro.azu.nl>, Henk Veldman
<H.Veldman-2 at neuro.azu.nl> wrote:

>David Micklem schreef:
>> Hi all,
>> Tetramethylbenzidine (TMB) is often used as a substrate for
>> horseradish peroxidase in ELISA assays, where the water soluble colour
>> is an advantage.
>> I notice that several companies now sell stabilised TMB substrate kits
>> that include an additive that causes the end-product to precipitate,
>> so that TMB can be used for staining nitrocellulose membranes etc.
>> Does anyone have a recipe for a precipitating version of the TMB
>> substrate mix?  I don't mind making it up fresh each time, so I don't
>> need the extra-stabilised ready-to-use, just pour it in, keeps for
>> months commercial version...
> - McKimm-Breschkin; J Imm Methods 135: 277ff (1990).

Perfect! Thank you Henk.

For future reference if anyone is interested, the protocol is:

Wash membrane in PBS after incubating with HRP conjugate.
Equilabrate in 10mM sodium citrate, 10mM sodium EDTA pH5.0 for 5 mins

Equilabrate in above buffer plus 1% dextran sulphate for 10 mins (this
is the crucial step).

Wash 3 times in citrate:EDTA buffer

Develop with 0.05mg/ml TMB in citrate:EDTA buffer



D.R. Micklem,              Time flies like an arrow... 
Dept. of Genetics,          Fruit flies like a banana.
Cambridge University,      Email:drm21 at mole dot bio dot cam dot ac dot uk. 
Cambridge, UK              Phone: +44 (1223) 766336
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