The world s first luminescent color development marker pen

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1 Reagent LuminolPenTM HRP system LuminolPenTM HRP system can provide the best way using luminescent to indicate target(s) while performing western blotting followed by luminescent color development..characteristics and advantages The world s first luminescent color development marker pen Innovated to provide the best way using luminescent for target(s) indication directly on the X-ray film. Calibration of molecular weight using chemiluminescent detection In chemiluminescent detection, LuminolPenTM HRP system marks the prestained marker position, which can be viewed on the film or the CCD imaging acquisition system. Labeler and annotation LuminolPenTM HRP system can be used on the transfer membrane to detect molecular weight markers and also provide labeling of samples and sample annotations, which can be displayed on the film. ECL reagent test indicator LuminolPenTM HRP system can indicate whether the ECL reagents are functional or not. 2.Introduction 2. Continuous evolution of immunoblotting Immunoblotting is also known as western blotting, a sensitive detector of specific proteins. Proteins are separated by a SDS-PAGE. The proteins are then transferred from the gel onto a membrane and probing the membrane with antibody. Compared to ELISA immunoblotting requires several steps and is time consuming, however, the accuracy of the assay is relatively higher. Apart from being a sensitive protein detector, an additional protein maker (Standard Protein Marker or Prestained Marker) is used to determine the protein molecular weight. Therefore, when the antibody specificity is not high, the protein molecular weight marker can assist in the determination of the target protein. Since 978, western blot has continuously evolved from the selection of different membrane materials, transfer methods, various antibody probes to detection techniques. Western blotting sensitivity has also risen from ng to fg. For instance, nitrocellulose was commonly used for electroblotting; however, it showed better retention for DNA and RNA but not

2 LuminolPen TM HRP system proteins. PVDF (polyvinylidene fluoride) membrane was then introduced, the hydrophobic and high binding characteristics showed better protein retention. 2.2 Using Prestained Marker in western blotting to determine protein molecular size In western blotting, proteins are separated according to their molecular size, providing an alternative target protein determinacy. In the past, to determine the position of target protein a standard protein marker would be loaded into the gel, followed by western blotting. The standard protein marker will then be subjected to CBR staining and compared with the chromogen treated target protein. Although this is a viable method, however, the steps are numerous. In 99 Shia proposed the usage of prestained marker as a more effective way to determine molecular weight of a protein, which does not require staining. Prestained marker is a range of protein covalently coupled to dye, which can be visualized on the membrane after electrotransfer. Colormetric/chromogen treated target protein can be directly compared to the protein marker. (Fig. A) Different from the traditional method of subjecting the standard protein marker to staining then compared with the colormetric/chromogen treated target protein. Prestained Marker Prestained Marker Target protein Filter Gel Membrane Filter Electrophoresis Transfer Membrane After trasfer membrane Get protein size directly Fig. A. Using prestained marker to determine protein molecular weight. Sample and marker loaded into the gel for electrophoresis followed by electrotransfer. Prestained marker can be visualized on the transfer membrane without further staining. The membrane is then subjected to immunoblotting and colormetric/chromogen treatment to reveal target protein, the protein size can be directly compared using the prestained marker. 2.3 Chemiluminescence posing difficulties on Prestained Marker reading In 888 Wiedemann introduced luminescence and categorized in to different types such as electroluminescence, thermoluminescence and chemoluminescence. Western blotting began to use chemoluminescence in place of c treatment. The utilization of chemiluminescent detection can enhance sensitivity leading to detection of proteins at an ng level. For further chemiluminescent information please refer to VisGlow. Prior to 2000, chemiluminescent reagent was relatively costly. Afterwards, it began to be used widely in chemiluminescent detection due to its high sensitivity in protein detection.

3 Reagent Although chemiluminescence development enhances sensitivity, however, usage with prestained marker bears a problem. In Fig. B, using the conventional prestained marker method, the marker can be directly visualized on the membrane. If subjected to chemiluminescent detection, the emitted light is captured through X-ray film or a CCD imaging acquisition system. Prestained marker cannot be detected by antibodies therefore will not be visible on the X-ray film or the CCD imaging acquisition system. This will result in faulty reading of target protein molecular weight. Colormetric/chromogen detection Chemiluminescent detection Prestained Marker Target protein Prestained Marker Target protein Transfer membrane + Image Transfer membrane Image Fig. B Prestained marker visualization using colormetric/chromogen and chemiluminescent detection. The prestained marker can be directly visualized on the transfer membrane using colormetric/chromogen method; in using chemiluminescent detection, the target protein is captured on an X-ray film whereas the prestained marker is not. Therefore the protein size cannot be determined directly on the X-ray film. In western blotting using colormetric/chromogen method, the protein size can be directly compared to the prestained marker on the membrane. Whereas using the chemiluminescent detection this is not possible, therefore the accuracy to determine the target protein decreases. 2.4 Present usage of chemiluminescent detection to determine protein size To over this difficulty there are products on the market. For instance, using a CCD imaging acquisition system. Fig. C, obtaining an image under normal light and an image using luminescence, using a computer the two images can then be merged and the marker can be directly applied to determine the protein size. However, the CCD imaging system is less sensitive as to the X-ray film and costly, this method is not widely practiced.

4 LuminolPen TM HRP system Prestained Marker CCD Imaging acquisition sysytem Capture Normal Image Target Protein Merged Image Capture luminescence Image Fig. C Image merges using the CCD imaging acquisition system. Obtaining an image under normal light and an image using luminescence, using a computer the two images can then be merged and the marker can be directly applied to determine the protein size. For laboratories not equipped with CCD imaging system or X-ray film development system other method can be applied. Fig. D. An estimation of protein size using superimposed image of the chromogen membrane and the X-ray film. This method is relatively inaccurate as it does not have a datum point. Prestained Marker Target protein Transfer Membrane X-ray Film Overlapping Difficult Fig. D. Using transfer membrane and X-ray film to estimate the protein molecular weight. Using superimposed image of the chromogen membrane and the X-ray film. This method is relatively inaccurate as it does not have a datum point.

5 Reagent Alternatively, users can chose to use the ECL TM DualVue Western Blotting Markers by GE. The western blotting markers are specifically detected by HRP conjugate and developed using chemiluminescent substrates on X-ray film or CCD imaging acquisition system. Due to the high cost of the kit, it is not widely used. 2.5 Chemiluminescent indirectly affects the outcome results of western blot. As stated in the previous section, there are ways to overcome molecular weight calibration using chemiluminescent detection. Using CCD imaging acquisition system or ECL TM DualVue Western Blotting Markers can accurately calibrate protein weight; however, the equipments and reagents are costly. In addition, CCD imaging acquisition system is less sensitive compared to X-ray film, therefore the film is more widely used. The disadvantage of using the X-ray film is that the prestained marker cannot be used for molecular weight calibration. For example, in Fig. E, during western blotting if the antibody specificity is not high, this will leading to non-specific binding and unable to determine which is the target protein. Molecular weight calibration must then be used to determine the target. Prestained marker can be directly visualized on the transfer membrane using colormetric/chromogen method. Chemiluminescent detection where using superimposed image of the transfer membrane and the film will not give an accurate reading due to this method does not have a datum point. Faulty reading can affect the development of a project, needless to say a great deal of time and money wasted and resulting in delay of publications. (A) (KDa) Target Protein (B) Up Down? Colormetric/chromogen detection Chemiluminescent detection Fig. E Calibration of molecular weight using chemiluminescent detection. Note: western blotting can result in binding of protein other than the target protein. (A) Prestained marker can be directly visualized on the transfer membrane using colormetric/chromogen method. (B) Chemiluminescent detection where using superimposed image of the transfer membrane and the film will not give an accurate reading due to this method does not have a datum point. When submitting an article with western blot data, the determinacy of target protein molecular weight is of importance. Reviewers will require authors to address this issue with direct evidence. However, to present there is still not an effective and direct method to demonstrate molecular weight calibration using chemiluminescent detection. Therefore,

6 LuminolPen TM HRP system often results are questioned and the articles may be rejected. 2.6 LuminolPen TM HRP system provides the solution To address the problem of molecular weight calibration using chemiluminescent detection, VisualProtein has developed a specific product- LuminolPen TM HRP system, the usage of LuminolPen TM HRP system is relatively simple. Following gel transfer, apply LuminolPen TM onto the prestained marker and proceed to immunoblotting. Areas marked by LuminolPen TM will react with chemiluminescent reagents and emit luminescence. The target protein and prestained marker will both appear on the film. Fig. F. Target protein and prestained marker appear on the film and molecular weight can be directly calibrated. (A) LuminolPen Filter Gel Membrane Filter Prestain Marker Eletrophoresis Gel Transfer Labeling React with antibody (B) General chemiluminescent detection Prestained Marker After using LuminolPen Fig. F Chemiluminescent detection of LuminolPen. (A) Following gel transfer, apply LuminolPen onto the prestained marker, specify date of experiment and add annotations then proceed to immunoblotting. (B) Chemiluminescent detection without applying LuminolPen, only target protein is revealed on the film, prestained marker does not appear on the film therefore molecular weight cannot be calibrated. Characteristics and advantages of LuminolPenTM HPR system recongnizes as follows The world s first luminescent color development marker pen Innovated to provide the best way using luminescent for target(s) indication directly on the X-ray film. Detection of molecular weight using chemiluminescent detection In chemiluminescent detection, LuminolPenTM HRP system marks the prestained marker position, which can be viewed on the film or the CCD imaging acquisition system. Prevent erroneous molecular weight calibration.

7 Reagent Labeler and annotation LuminolPen TM HRP system can be used on the transfer membrane to detect molecular weight markers and also provide labeling of samples and sample annotations (Fig. G), which can be displayed on the film. Prestained Marker Sample No. Exp. Date Fig. G LuminolPen to label and annotation results. LuminolPen marks molecular weight markers, provide labeling of samples and sample annotations for future reference. ECL reagent test indicator LuminolPen TM HRP system can indicate whether the ECL reagents are functional or not. Many factors can lead to no images appearing on the film. If areas marked by LuminolPen simultaneously does not appear on the film, this indicates the ECL reagent is no longer functional (Fig. H). (A) (B) (C) Fig. H Using LuminolPen as an ECL reagent functionality indicator. (A) Appearance of prestained marker marked by LuminolPen and target protein, indicating the ECL reagent is functional. (B) Appearance of prestained marker marked by LuminolPen, however, the target protein does not appear. Indicating the protein amount may be no detectable. (C) Both prestained marker marked by LuminolPen and target protein does not appear, indicating the ECL reagent has no function.

8 LuminolPen TM HRP system 3.Method Application of LuminolPen TM HRP system Apply LuminolPen following transfer of PVDF or nitrocellulose membrane. Application of LuminolPenTM HRP system.remove membrane and place it on a filter sheet. 2.Remove excess solution using a filter sheet where LuminolPen will mark. Membrane place on a filter NOTE:.Remove excess solution, however, keep membrane in a moist state. 2.Remove excess solution to allow LuminolPen reagent to bind onto the membrane. The binding of LuminolPen reagent will affect the brightness on the film. TIPS: Place a piece of filter paper where prestained marker is, gently press to absorb excess solution and carefully remove filter paper. Remove excess solution by filter sheet 3.Remove LuminolPen from the refrigerator, mark on prestained marker several times according to supplement 8.. NOTE:.Different blocking buffer affect the brightness of LuminolPen on the film or CCD imager. Refer to supplement 8. for the number of markings. Marked by luminopen 2.If marking resistance occurs during marking, moist the tip of LuminolPen will solve the problem. 4.After LuminolPen marking, place the membrance in blocking solution and proceed to immunoblotting. Blocking membrane NOTE:Mark using LuminolPen prior to membrane blocking, otherwise the signal emitted by LuminolPen will decrease. 4.Troubleshooting Weak signal Problems Cause. Usage after blocking 2. Excess solution on membrane 3. Presence of sodium azide Solution. Use prior to blocking 2. Remove excess solution from membrane 3. Mark several more times Signal too strong Strong antibody signal Decrease the number of LuminolPen marking Resistance during markings Dry pen tip Moist pen tip

9 Reagent 5.Application note Wear protective clothing and gloves while applying LuminolPen. Prevent direct skin contact with reagents from LuminolPen. Flush with large quantity of water if exposed to reagent. 6.Storage Store LuminolPen TM HRP system in 4 o C. Use within 6 months after opening. 7.Item LuminolPen TM HRP system (LH603) LuminolPen User manual LH603 pen booklet 8. Supplement 8. Suggestion on the number of time to mark LuminolPen TM HRP system according to various blocking buffer Blocking Buffer Exposure Time 0.5mins mins 5mins 30mins hrs Skim Milk BSA Casein