Alkaline Phosphatase Substrates: Ready to Use – ATTOGLOWTM
Available Sizes100mL
  250mL
 500mL
  1000mL
  1000mL

  Application
SAP450101Alkaline Phosphatase Substrate with Enhancer-450 in bufferImmunoassays, Reporter gene assays
SAP540102Alkaline Phosphatase Substrate with Enhancer-540 in bufferQuantitative PCR, Blotting
SAP450103Alkaline Phosphatase Substrate with Enhancer-450 in buffer LB(PVDF & Nitrocellulose Membrane)
SAP540104Alkaline Phosphatase Substrate with Enhancer-540 in buffer LB 
SAP450105Alkaline Phosphatase Substrate with Enhancer-450 in buffer PLUS 
SAP540106Alkaline Phosphatase Substrate with Enhancer-540 in buffer PLUSNylon Membrane
SAP450107Alkaline Phosphatase Substrate with Enhancer-450 in buffer NM 
SAP540108 Alkaline Phosphatase Substrate with Enhancer-540 in buffer NMNylon Membrane
SAP450111 Alkaline Phosphatase Substrate with Enhancer-450 in buffer LBNMNylon Membrane
  LB – Low Background, NM – Nylon Membrane 
NEW PRODUCT  
SAP450103Alkaline Phosphatase Substrate with Enhancer-450 in buffer 
SAP540104Alkaline Phosphatase Substrate with Enhancer-540 in buffer 

Alkaline Phosphatase Substrates without Enhancer 
SAP11111X, Alkaline Phosphatase Substrate in buffer
SAP1011210X,Alkaline Phosphatase Substrate in buffer
SAP11131X, Alkaline Phosphatase Substrate in buffer LB
SAP1011410X, Alkaline Phosphatase Substrate in buffer LB
SAP11151X, Alkaline Phosphatase Substrate in buffer PLUS
SAP10116 10X, Alkaline Phosphatase Substrate in buffer PLUS
SAP11171X, Alkaline Phosphatase Substrate in buffer NM
SAP111710X, Alkaline Phosphatase Substrate in buffer NM
  
NEW PRODUCT 
SAP21132X, Alkaline Phosphatase Substrate in buffer LB

DIRECTIONSChemiluminescent Substrate for Alkaline Phosphatase Enzyme
1. Store the reagent bottle at 2-6o C
2. Equilibrate at room temperature for 30 minutes before use.
3.Do not contaminate the substrate with any phosphate buffer or alkaline phosphatase enzyme.
4.Use only toxin free BSA as a blocking reagent for micro titer plates or membrane to lower the background luminescence.
5. Quality of BSA can be checked by alkaline phosphatase (AP) enzyme substrate. Transfer 100 ml of substrate in a tube or micro titer plate and record the background luminescence. Now add 10 ml of 0.1% BSA in tris buffer, pH 8.0 to 8.5, to 100 m of BSA is excellent.
6. The chemiluminescence substrate for the alkaline phosphatase enzyme is an extremely sensitive reagent, and must be protected from extraneous sources of alkaline phosphatase, including that produced by exposure to environmental bacteria and pipettors that may have come in contact with AP enzyme, AP conjugated antibodies or AP conjugated streptavidin. When setting up an experiment, aliquot the required quantity of reagent by pouring out of the original bottle into a clean, alkaline phosphatase free container. Discard any excess reagent. Do not return excess material to the stock reagent bottle.
7. Alkaline phosphatase enzyme substrate has a wide range to detect alkaline phosphatase enzyme in solution as well as on membrane. The lower enzyme concentration may be a few atto grams and the highest concentration alkaline phosphatase enzyme may be a few nano grams depending on the source of the alkaline phosphatase enzyme.
8. For good results, wash your tube or micro titer plate or membrane with 0.2M tris buffer, pH 9.5 to 9.7, and then use the substrate.
9.Best results for chemiluminescence can be obtained from 15 minutes to 120 minutes a fter contacting the substrate with alkaline phosphatase enzyme.
10.Alkaline phosphatase substrate is sensitive to light. The product is provided in an plastic bottle, and if storage outside of the original plastic container is required, wrap aliquots with aluminum foil.

Instructions for Blotting with Alkaline Phosphatase Substrates


 Streptavidin-AP Conjugates
1. Separate proteins by electrophoresis.
2. Transfeer proteins to the membrane.
3.Block the membrane with blocking buffer (check blocking buffer with AP substrate for background).
4. Incubate the membrane with primary antibody for 30 to 45 minutes.
5.Wash the membrane with washing buffer.
 Incubate the membrane with biotin labeled secondary antibody for 30 to 45 minutes.
6.Wash the membrane with washing buffer.
7.Incubate the membrane with Streptavidin-AP conjugate (diluted in MDPvt.Ltdenzyme diluent or other source) 30 to 45 minutes.
8.Wash the membrane with washing buffer (MDPvt.Ltdwash buffer or other source) 5 to 6 times.
9.Incubate the membrane with enzyme substrate for 2 to 3 minutes.
10.Drain the excess substrate from the membrane.
11.Wrap the membrane in a plastic.
12. Expose the membrane to film or take a picture with CCD camera

 Secondary Antibody-AP Conjugate
1.Separate proteins by electrophoresis.
2.Transfer proteins to the membrane.
3.Block the membrane with blocking buffer.
4.Incubate the membrane with primary antibody for 30 to 45 minutes.
5.Wash the membrane with washing buffer.
6.Incubate the membrane with AP labeled secondary antibody for 30 to 45 minutes.
7. Wash the membrane with washing buffer.
8.Wash the membrane with washing buffer (MDPvt.Ltdwash buffer or other source) 5 to 6 times.
9.Incubate the membrane with enzyme substrate for 2 to 3 minutes.
10.Drain the excess substrate from the membrane.
11.Wrap the membrane in a plastic.
12. Expose the membrane to film or take a picture with CCD camera.
 Control No.: IMDPvt.Ltd-01, Rev. 1

Enzyme Immunoassay Procedure (General Method):


 Coating plates
1.Wash the plate with washing buffer.
2.Block the remaining active sites of the plate or tube with blocking buffer (check the blocking buffer with AP substrate for background).
3.Wash the plate or tube with washing coat microtiter plate or tube with antibodies* or antigen*.
4.Buffer and dry at room temperature or room temperature under vacuum and store properly.

 Assays for haptens (anti-hapten antibodies coated plates):
1.Add the diluted antigen-enzyme conjugate to the plate with different concentration of antigen and incubate at room temperature or 37oC for 30 minutes to 120 minutes.
2. Wash the plate with washing buffer at least three times. In assays, if the enzyme is alkaline phosphatase, washing buffer should be 0.2M tris-buffer, pH 7.2 to 7.5.
3.Add the enzyme substrate to the plate and incubate at room temperature or 37oC for 30 minutes to 120 minutes.
4.Read the plate.

 Assays for proteins (anti-protein antibodies coated plates):
1. Add the different concentration of antigen protein diluted in buffer to the plate and incubate for 30 to 120 minutes.
2. Wash the plate three times with washing buffer.
3.Add the anti-protein antibody (monoclonal)-enzyme conjugate diluted in enzyme diluent buffer and incubate 30 to 120 minutes.
4.Wash the plate at least three times with washing buffer. In assays if the enzyme is alkaline phosphatase, washing buffer should be 0.2M tris-buffer, pH 7.2 to 7.5.
5. Add the enzyme substrates and incubate at room temperature or 37oC for 30 to 120 minutes.
6. Read the plate.
 Advantages of using chemiluminescent assays:
Chemiluminescent assays are highly sensitive. For coating the plate the amount of antibody should be reduced at least 10 times less compared to colorimetric assays. Likewise, the concentration of enzyme conjugate can be markedly decreased.
Chemiluminescent assays are highly reproducible.
Chemiluminescent assays have no edging effects.
Chemiluminescent assays do not need stopping reagents.
Chemiluminescent assays have a wide range of detection.
Control No.: IMDPvt.Ltd-03, Rev. 0

Chemiluminescent Substrate Alkaline Phosphatase Enzyme


Alkaline phosphatases (orthophosphoric monoester phosphohydrolase, alkaline optimum) are found primarily in animal tissues and microorganism. Alkaline phosphatases used in EIA are isolated from bovine intestinal mucosa or from E. coli. These enzymes have considerable differences in their properties and should not be assayed under identical conditions. The bacterial enzyme has lower activity than the bovine intestinal enzyme.

Alkaline phosphatases hydrolyze numerous esters, such as those of primary and secondary alcohols, phenols and amines. A major reason for the popularity of alkaline phosphatase for EIA is its absence from higher plants. The enzyme is abundant in animals and human tissues involved in nutrient transport and in developing tissues and secretor organs, but it is not found in significant amounts in muscle, connective tissue or cartilage. Some pathological conditions increase alkaline phosphatase activities in sera.

The enzyme transfers the phosphoryl residue via a phosphoryl-enzyme intermediate, which can be repressed by inorganic phosphate. The Comparative detection limit of alkaline phosphatase using fluorescence, time-resolved fluorescence and colorimetric techniques are 10-19 M (6×104 molecules), 3×10-19 M (1.8×105 molecules) and 5×10-17 M (3x108molecules), respectively. Stabilized 1, 2-dioxetane substrates* provide high signal, low background, wide dynamic range, rapid results and excellent reproducibility. These 1, 2-dioxetanes provide substrates which are highly sensitive but can detect an enzyme concentration up to 10-21M (6X102 molecules of alkaline phosphatase) in solution as well as on a membrane (best commercially available).

Our new series of ultra-sensitive 1, 2-dioxetanes can detect alkaline phosphatase at attogram level. MDPvt.Ltdchemiluminsecent system is at least 100 times more sensitive compared to the best competitor. These novel 1, 2-dioxetanes are protected by US Patent 6,461,876B1 and other pending PCT and US patent applications. The kinetic results of low level of alkaline phosphatase enzyme and MDPvt.Ltdsubstrate can be shown as:


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MDPvt.Ltdsubstrates for alkaline phosphatase are more sensitive due to the effect of -electrons on the decomposition of 1,2-dioxetanes intermediate in the form of phenoxide ion formed after the interaction of phosphate group on 1,2-dioxetane and alkaline phosphatase enzyme and can be shown as:

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MDPvt.Ltdoffers a number of kits and substrates that are designed specifically to alkaline phosphatase enzyme. Our line of chemiluminescent alkaline phosphatase enzyme substrates includes phosphate derivatives of several different 1, 2-dioxetanes.

* Our 1, 2-dioxetanes are exlusively supplied by Life Technologies in the USA and Canada.