Technical Data Sheet. Sensido Plus ECL Substrate (HRP) Description. Advantages

Transcription

1 ECL Substrate (HRP) Catalog R13-VG08 Technical Data Sheet ADD: 7F, 72, Song-Te Rd., Taipei 110, Taiwan TEL : Package Cat. Components Solution A (Substrate Solution) Solution B (Peroxide Solution) Volume Package R13-VG R13-VG ml each 250ml each 2 bottle 2 bottle Storage Stored at 4 C and shielded from light. Please use up the product in 12 months. Description ECL Substrate is an advanced ECL product in terms of efficacy and sensitivity for chemiluminescent detection of immobilized proteins (Western blotting), conjugated with Horseradish Peroxidase (HRP) directly or indirectly. ECL Substrate is at least 8 fold more sensitive than other commercial products (Figure 1 and Figure 2). Employing ECL Substrate, the chemiluminescence emission can retain more than 50% of its original intensity after 90 minutes reaction(figure 3). Owing to the strong signal, very short exposure time to the X-ray film or in other documentation systems is required when using Sensido Plus ECL Substrate in the Western blotting experiments. This feature benefits the researchers with excellent low background results (Figure 4). Advantages 1.High sensitivity Detect target protein at pg level (Figure 1 and 2). 2.Lasting duration Retain up to 78% of its maximal intensity after 60 minutes reaction (Figure 3). 3.Antibody saving Generate strong signal with minimal usage of antibody (Figure 4). 4.Low background Reveal your result with insignificant background and less non-specific signals

2 Instruction A. Protein Transfer 1.Perform 1D or 2D electrophoresis for protein separation. 2.Move the electrophoretic gel into appropriate transfer buffer and equilibrate for 10 minutes. 3.Wet the PVDF or nitrocellulose membrane in transfer buffer. (For PVDF membrane, it is necessary to pre-wet it in methanol before moving into transfer buffer). 4.Assemble the transferring sandwich as the order of two filter papers, gel, membrane and two filter papers. 5.Transfer proteins according to blotting apparatus manufacturer s instruction. 6.Wash the transferred membrane of three times by Wash buffer for 5 minutes. B. Antibody Incubation 1.Add Blocking buffer and incubate at room temperature for 30 minutes. 2.Prepare the primary antibody by diluting it with Blocking buffer according to the manufacturer s instruction or previous experience. (Due to the good sensitivity of chemiluminenscent detection, previous antibody dilution factor can be increased 2-5 folds for optimal signal to noise ratio.) 3.Add primary antibody and incubate at room temperature for at least 1 hour with gentle agitation. For more specific interaction between primary and antigen proteins, it is recommended to perform additional incubation at 4ºC 8-12 hours. 4.Decant the primary antibody solution thoroughly. Wash the membrane of at least three times with ample amount of fresh Wash buffer for 10 minutes. 5.Prepare the secondary antibody by diluting it with Blocking buffer according to the manufacturer s instruction. (Due to the good sensitivity of chemiluminenscent detection, secondary antibody dilution can start from 1:20000) 6.Add secondary antibody and incubate at room temperature for 1 hour with gentle agitation. 7.Decant the secondary antibody solution thoroughly. Wash the membrane of at least four times with ample amount of fresh Wash buffer for 10 minutes

3 Instruction C. Chemiluminenscent Detection 1.To prepare working HRP substrate, mix equal volume of Solution A and Solution B in a clean tube freshly. 0.1 ml of working HRP substrate is sufficient per 1 cm 2 membrane area. 2.Allow the HRP working substrate to stand at room temperature for 3 minutes with proper light shielding. 3.In the dark room or box, place the membrane side up in a clean box or plastic wrap. Add the HRP working substrate onto the membrane. 4.Incubate the membrane at room temperature for 10 seconds. 5.Overlay plastic wrap or a transparency sheet on the wet membrane. NOTE: Keep the membrane wet while exposing!! (see next step) 6.Expose the membrane to appropriate X-ray film or by chemiluminenscent image acquisition system. It is recommended to use 5 seconds as the initial exposure time. 1 minute exposure usually delivers stronger signal with insignificant background. D. Striping of PVDF Membrane The immunoblot of PVDF membrane can be stripped of antibodies, and then reprobed. 1.Incubate membrane in stripping buffer (62.5mM Tris-HCl ph 6.8, 100mM b-mercaptoethanol and 2% (w/v) SDS) for 30 minutes at C. 2.Wash the membrane twice in Wash buffer for 10 minutes each. 3.To ensure complete removal of antibodies, incubate the membrane with ECL Substrate working substrate and expose against X-ray film for 5 minutes. No signal should be observed for complete stripping. Safety Information Other Reagents Needed for Western Blot Please wear gloves, lab coat and gaggles while operating. Prevent contact product directly. In case of contacting, wash with large amount of water. 1.PVDF or nitrocellulose membrane. 2.Wash buffer: Phosphate-buffered saline (PBS) or Tris-buffered saline (TBS) containing % Tween -20. PBS: 10 mm sodium phosphate, 150 mm NaCl, ph 7.2 TBS: 10 mm Tris, 150 mm NaCl, ph Blocking buffer: 1 5% (w/v) blocking agent (e.g., casein, BSA, or gelatin) in wash buffer. 4.Specific primary antibody for interested protein, diluted in blocking buffer. 5.HRP-conjugated secondary antibody, specific for primary antibody, diluted in blocking buffer. 6.X-ray film or chemiluminescence image acquisition systems

4 Trouble Shooting Possible cause Technical Data Sheet Remedy Problem 1: No signal or weak signal 1. Poor transfer efficiency 1. Optimize the membrane transferring procedure. 2. Insufficient antigen 2.1. Increase the amount of loaded antigen. 2.2 Make sure the blot have been store correctly to avoid the degradation of target protein. 3. The concentration of primary and secondary antibody is too low 4. Inappropriate storage/preparation of the ECL detection reagents 5. Too short exposure time 3. Increase the concentration of the primary and/or the secondary antibody. 4. Use HRP or HRP conjugates to check the applicability of ECL reagents. 5. Extend exposure time Problems 2: Excessive signal 1. Antigen or antibody excess 1.1. Reduce the amount of loaded antigen Dilute the primary antibody and/or the secondary antibody. Problem 3: High Background 1.Antigen or antibody excess 1. Optimize the condition by reducing the amount of antigen, or the concentration of the primary antibody and/or secondary antibody. Initially, reduce the secondary antibody to 20% of the original usage. 2.Inappropriate blocking 2. Try different blocking substrate such as gelatin, casein, skim milk or casein. 3. Inadequate washing 3.1. Increase the concentration of Tween-20 in washing solution Increase the washing steps between the hybridization procedures Extend washing time. 4. Overexposure to film 3.1. Shorten the exposure time.

5 HRP (pg) Ab-HRP (pg) Figure 1: The comparison of sensitivity for ECL Substrate, brand P Plus and brand A ECL. Two fold serially diluted HRP from 1000 to 8 pg was used for evaluation. After adding the ECL substrates for 5 minutes, the signals were recorded by exposing to an X-ray film for 5 seconds. The arrows indicate the samples with comparable intensity. It can be found that the ECL signal from ECL Substrate is 8 fold stronger than from other brands. 12x pg ALU (absolute luminenscent units) 10x10 6 8x10 6 6x10 6 4x10 6 2x HRP (pg) Figure 2: Densitometric analysis of the result in Figure 1 from 0 to 125 pg of HRP. The ECL signal from ECL Substrate is 8 fold stronger than from other brands

6 12x10 6 ALU (absolute luminenscent units) 10x10 6 8x10 6 6x10 6 4x10 6 2x % of signal 78% 0 10% 12% time ( minutes) Figure 3: The duration test for ECL Substrate, brand P Plus and brand A ECL. After 60 minute reaction, the chemiluminenscence emission from ECL Substrate retains up to 78% of its original intensity while other brands retain only 10-12%. Human plasma (1X = 1mL) 1X 1/3 1/9 1/27 1/81 1/273 Sensido Recenttec Plus Human serotransferrin 1st Ab : mouse IgG anti human serotransferrin (1:20000) 2ndAb : rabbit anti mouse IgG HRP (1: 60000) 5 seconds exposure on X-ray film Figure 4: The comparison of WB application for ECL Substrate, brand P Plus and brand A ECL. Human plasma with 3-fold serial dilution from 1mL was separated by 12.5% SDS-PAGE and probed by anti human serotransferrin. All results were exposed to X-ray film for 5 seconds