Paul.xu at 23:00, 11 February 2014

2014-02-11T23:00:24Z

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== Reagents ==

Peroxidase from horseradish type VI-A, RZ ap. 3.0 (Sigma) was prepared in Tris-HCl buffer 0.1 M pH 8.5. Luminol (5-amino-2,3-dihydrophthalizine-1,4-dione) (Sigma) 0.01 M was prepared by dissolving 0.0913 g of luminol (97%) in a small amount of NaOH solution, adding Tris-HCl buffer 0.1 M (pH 8.5) until the Ænal volume was 50 ml. Hydrogen peroxide 33.3% (Probus) was prepared in bidistilled water to give a final concentration of 0.1 M. p-Iodophenol 99% (Aldrich), p-coumaric acid (Sigma) and 2-naphthol (Sigma) were prepared in bidistilled water to obtain a final concentration of 0.001, 0.001 and 0.01 M, respectively. A little volume of dimethyl sulfoxide is added to dissolve p-coumaric acid and 2-naphthol. 

 

1. '''Luminol '''(C8H7N3O2, 5-amino-2,3-dihydro-1,4-phthalazinedione, also known as 3-aminophthalhydrazide), ≥97% HPLC (cat. no. A8511, Sigma-Aldrich, St. Louis, MO); 

 250 mM luminol prepared in dimethyl sulfoxide (DMSO), stored in ~55 μl aliquots at -20°C 

443 mg in 10 ml DMSO--> 250 mM.

 2. '''p-Coumaric acid''', ≥98% HPLC (cat. no. C9008, SigmaAldrich); 

(para-coumaric acid, 4-hydroxycinnamic acid, β-(4-hydroxyphenyl)acrylic acid) 

90 mM p-coumaric prepared in DMSO, stored in ~25 μl aliquots at -20°C 

150 mg in 10 ml DMSO-->90 mM

 3. '''H2O2''' solution, 30% (w/v) (cat. no. 216763, Sigma-Aldrich) 

 4. DMSO, ≥99.5% (cat. no. D5879, Sigma-Aldrich) 

 5. ECL buffer; 0.1 M Tris pH 8.6 at 22°C, stored at 4°C, 

4.5 g Tris-HCl, 2.65 g Tris-base, ddH2O to 500 ml. 

''' '''

=== '''Two Solutions Method:''' ===

'''Solution 1''' contains 780 ug ml-1 of luminol and 950 ug ml-1 of PIP of in 0.1M Tris-HCl, pH 9.35. 

'''Solution 2''' contains 100 ul 30% hydrogen peroxide in 1 l of 0.1M Tris-HCl, pH 9.35. 

Just before use, equal volumes of each solution are mixed together. 

A cocktail composed of 1 ml luminol (4mg ml-1 in DMSO), 1 ml p-iodophenol (1mg ml-1 in DMSO), 0.6 ml of 1M Tris-HCl (pH 7.5), 5 ul of 30% H2O2, and 7.5 ml dH2O works equally as well. 

=== '''Two Solutions Method 2''' ===

Take two containers (they need to be something with a secure lid and which you can keep out of the light) and label one ‘A’ and one ‘B’.

In the '''first tube (A)''', combine the following:

1 ml luminol solution 0.44 ml coumaric acid solution 10 ml Tris-HCl 1M, pH 8.5 Distilled water up to a final volume of 100 ml

In the '''second tube (B)''', combine the following:

64 µl hydrogen peroxide 10 ml Tris-HCl 1M, pH 8.5 Distilled water up to a final volume of 100 ml

'''STORAGE''': Keep both solutions in the dark at 4°C. I used to wrap my solutions in aluminium foil to keep the light away from them, or keep them in a dark-coloured glass flask.

'''SHELF LIFE''': The solution generally lasts up to 1-2 months if stored correctly. If you’re concerned that it’s been sitting in the fridge for too long, it’s probably best to just make more.

NOTE: You can easily halve or quarter the volumes of solutions described here if you want less than 100 ml each of solutions A and B – if your lab only does western blots sporadically, this may be useful to you, as it’ll waste less solution if it goes off in between experiments. 

=== Two Solution Method 3 ===

'''Stock solution'''

10 mM Luminol (MW177.2) in 50 % DMSO: 21.24 mg/12 ml Dissolve luminol in 6ml 100% DMSO then dilute to 50% DMSO

50 mM p-iodophenol (MW 220.01) in 50 % DMSO: 0.55 g/50 ml

 

'''Solution A''' 100mM glycine pH 10 (with NaOH) 0.4mM luminol 8 mM 4-iodophenol

 

Glycine 0.751 g 10 mM Luminol 4 ml 50 mM 4-iodophenol 16 ml DW (total 100 ml) 80 ml Keep in a brown bottle wraped with aluminium foil at 4°C.

 

'''Solution B''' 0.12% (w/w) hydrogen peroxide in water 30% H2O2 400 µl/100 ml Keep at 4°C.Both solutions will keep at 4°C for at least a year.

 '''Usage'''

Mix equal volumes of solution A and solution B. Incubate the blot in developing solution for 1 minute. Drain excess liquid and wrap blot in plastic film. Expose to film. 

== Usage ==

Combine the following in chronological order while stirring: 

10 ml ECL buffer 

22 μl p-coumaric acid 

50 μl luminol and 3 μl H2O2. 

Pour onto membrane and allow to stand undisturbed for 90 sec.

Drain the membrane of ECL detection reagent, wrap the membrane in Saran Wrap or a sheet protector and expose. 

 

== Purification of p-iodophenol (PIP) and luminol ==

PIP is needed for enhancing the visible light reaction by acting as a co-factor for peroxide activity toward luminol, rather than participating directly in the luminescence itself. When phenolic enhancers are used in combination with HRP, the level of light is said to be increased about 100-fold. This is why the reaction has been dubbed `enhanced' chemiluminescence [2] or `ECL' (not to be confused with electrochemiluminescence, which has also been called by the same acronym ECL).

The cheapest PIP be purchased from Aldrich or Fluka and then sublimed in-house at 85 degrees C under vacuum. This is extremely easy to do and results in beautiful white crystals of pure iodophenol as compared to the mucky yellow powder obtained from the chemical supply company. the commercially available apparatus for this is  expensive, but that a cheaper homemade version can be made for little cost.

To prepare luminol, suspend 3 g of the cheapest luminol in 90 ml of 1-butanol and to add 40 ml of 1M NaOH. After the luminol is dissolved by warming and agitating, 30 ml of methanol is added and the recipient cooled in ice before adding 40 ml of 1M HCl. This should then be left at -20 degrees C for 30 min for precipitation. The crystals are then filtered onto a Whatman No. 1 filter, rinsed with dichloromethane and dried under vacuum. To protect the crystals from light, the bottle or tube should be wrapped with a piece of aluminum foil and placed in a dessicator. 

== Stripping blots ==

Most antibodies should be removed by heat and SDS:

100 mls

b-ME 100 mM 0.71 mls 14.1 M stock from bottle

SDS 2% 20 mls 10% stock

62.5 mM Tris, pH 6.8 12.5 mls SDS-PAGE upper gel buffer 

== Ref ==

1. http://www.ncbi.nlm.nih.gov/pubmed/17141265: 

4-iodophenylboronic acid (4IPBA) generated strong specific signals and low background chemiluminescence. We optimised the luminol, 4IPBA and hydrogenperoxide concentrations of this 4IPBA-ECL solution. The optimised 4IPBA-ECL solution (100 mM Tris/HCl pH 8.8, 1.25 mM luminol, 2 mM 4IPBA, 5.3 mM hydrogenperoxide) shows a greatly increased signal intensity compared to the initial pCA-ECL protocol and to some commercially available ECL solutions. In addition, the optimised 4IPBA-ECL solution also generates much lower background chemiluminescence than other non-commercial ECL solutions using p-coumaric acid or 4-iodophenol as enhancers. The 4IPBA-ECL solution was stable when stored but had the lowest background when prepared freshly from stock solutions. Thus, we present an optimised protocol for a well-performing inexpensive ECL solution which is an alternative to expensive commercial ECL solutions and which achieves a better signal and lower background than the commercial solutions tested. 

[[Category:Protocols]]

ECL - Revision history

Paul.xu at 23:00, 11 February 2014