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Resources by Application
A. Each type of kit has been validated for the sample types indicated in its protocol. All other sample types would have to be validated internally. Please contact ALPCO's product support team for further information regarding possible testing of other sample types performed by the product users.
A. Each type of kit has been validated for the species indicated in the protocol. Although cross reactivity may exist, validation of other species would need to be performed internally. Please contact ALPCO's product support team for further information regarding possible species validation performed by the product users.
A. Each kit will include at least one set of standards (or one vial of standard to be diluted) and in most cases one or two controls. Standards and controls (if included) must be run each time the assay is performed and it is highly recommended to run all standards/controls/and samples in duplicate.
A. No. Do not substitute components from other kits or lots.
A. No. Please do not deviate from the protocol.
A. No. The kit should not be used if it is past the expiration date specified on the kit label.
A. Please be sure that the kit and its components are stored as indicated in the kit insert. For additional information about kit stability at room temperature please contact ALPCO's product support team.
A. No. If a control(s) is provided always be sure to run the control(s) with each assay. The concentrations of the controls should fall within the range specified on the certificate of analysis.
A. We do not recommend the use of a multichannel pipette. Wash buffer must be dispensed with adequate and equal force to properly wash the wells. We recommend the use of a Statmatic wash nozzle www.tricontinent.com/products/statmatic-i/ or an automated plate washer. Please refer to the Learning Center tab of the Resources section of the website for a video displaying proper washing technique or click here.
A. It is extremely important to know where your standards, controls and samples are located on the plate so that results are generated appropriately. Click here for a downloadable platemap. It also helps to label the strips on the plate (use small tabs at each end) in the event that strips become loose while decanting.
A. Correct washing of the plate is a very critical and important aspect of running any successful ELISA. Consistency in washing the plate is essential. Washing the plate too rapidly or too slowly, incomplete washing or aspirating, and allowing the wells to sit dry are all factors that will affect the precision of the assay. We do not recommend the use of a multichannel pipette for this purpose. Multichannel pipettes do not apply enough pressure to thoroughly remove the unbound material and this will result in higher background noise. A video displaying proper washing technique is available in the Learning Center section of the website or click here.
A. Recommended freeze/thaw cycles vary based on kit. We recommend to avoid repeated freeze and thaw cycles unless otherwise indicated in the manual. If the collected sample amount allows for it, prepare and freeze aliquots to eliminate or reduce freeze/thaw cycles.
A. Each kit contains diluents and buffers that are formulated to closely match specific sample types. The manufacturer will not guarantee the performance of the kit if components that were not provided with the kit are used to run the assay.
A. Enough reagents are provided to run the entire kit when the protocol is followed. If your type of study requires a greater dilution, calculate the amount of diluent needed prior to setting up the assay and additional diluent may be available for purchase. Please contact customer support for information on the availability of individual components.
A. The concentration of the controls must be within the range specified in the certificate of analysis. If the controls are out of range the assay may be invalid. Also, make sure the curve fit recommended in the protocol has been used. If more than one recommendation is provided use the regression method that best fits the standard data points.
A. No. Never use example values to calculate your assay results. Sample concentrations should be generated from the standard/calibrator values obtained with each assay.
A. Only sample values that fall within the range of the standard curve can be used. Values outside of this range are generally not linear and can lead to incorrectly extrapolated results.
A. The sample may contain the analyte but it is undetectable based on sensitivity of the kit. The matrix of the sample may also be masking the detection. Ensure that the dilutions have been made as stated in protocol and review the procedure to ensure all reagents were added with the correct volume and in the correct order.
A. ALPCO now offers a tool to help with calculating the number of samples that can be run in a kit. Once you review the protocol to determine the number of standards, controls, and blanks that may be required for the specific kit you plan to run click here to go to the Sample Calculator.
A. A reference wavelength is a secondary wavelength used for correction (normalization) of the principal wavelength OD values. It helps to account for imperfections in the plate.
A. The measurement wavelength is determined by the substrate used in each kit. The most common one is TMB. It produces a blue color measurable at a wavelength of 650nm. It can be used in end point assays by stopping the reaction with either 1M phosphoric acid or 1M sulfuric acid. A yellow reaction product is formed upon acidification that is measurable at 450 nm.
A. The main difference between measure wavelength and reference is that the first one has been chosen to read the absorbance produced by the substrate and the second one (reference) to detect imperfections in the plate.
A. Yes, you can. However, the use of a reference wavelength may improve the sensitivity of the assay.
A. Fluorescence is the emission of light by a substance that has absorbed light. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed wavelength. In flow cytometry, fluorophores are used to tag antibodies in order to provide a signal upon detection of an antigen on the sample when exposed to a single wavelength laser.
A. There are several different methods that can be used to conjugate fluorophores to antibodies. This can depend on the antibody and type of fluorophore being used. One common method is to conjugate the organic fluorophore via primary amines (lysines) on the antibody. Another method conjugates maleimide-labeled fluorophore with antibody via sulfhydryl groups in the hinge region.
A. Isotype controls are recommended for every flow experiment. Isotype controls that do not recognize any known proteins will provide you with information on the background staining of an antibody due to its isotype. For multicolor experiments, fluorescence-minus-one (FMO) controls can help you determine the fluorescence spillover from all your other antibodies and can be used to help in gating positive and negative boundaries. Unstained cells can also provide you with a relative measure of the auto fluorescence associated with any particular cell type.
A. Refer to your instrument manual to determine the specifications of your instrument. You may also be able to find specifications in the software provided with your instrument. Note that many instruments are custom built and may not match the standard manual. Also, many instruments have adjustable filter sets, allowing you to configure your instrument to suitably run many different combinations of fluorophores. Always verify that the instrument you plan to use is capable of detecting the fluorophores in your experimental panel.
A. Compensation is the process of removing spillover (spectral overlap) from other fluorophores into the detector for your fluorophore of choice. For example, due to the wide emission range of FITC, some of the FITC signal "bleeds" into PE giving you false signal in the PE channel. On newer digital instruments, compensation can be applied automatically using single stain controls.
A. Fixation with paraformaldyde generally tends to decrease the fluorescence intensity of bound antibodies, particularly with prolonged exposure. This is especially true for nanocrystals and tandem dyes such as those derived from PE and APC. Excessive fixation can also alter the conformation of proteins, causing loss of antibody reactivity to proteins as well.
A. Tandem dyes are fluorophores composed of two distinct fluorophores conjugated together. The resulting tandem uses the excitation property of the donor fluorophore and emission property of the acceptor fluorophore, based on the principles of fluorescence resonance energy transfer (FRET).
A. I would always recommend performing a titration because sometimes you find you can use less antibody than what is recommended for your own experimental needs, and also to have a feel for how the antibody behaves in your own hands. The % positive staining should be maintained with concentration selected - i.e. if while they are titrating they find the population % drops then they are not staining all the cells and should use a higher concentration.
A. A functioning HPLC system includes a sampler, pump, column, detector and data processor. A degasser and column oven may also be used.
A. There are UV, fluorescence, electrochemical, conductivity, refractive index, evaporative light scattering, chiral, radioactive and mass spectrometry detectors.
A. Check the assay protocol to instructions on the appropriate column. The most commonly used column is C18 which covers a wide range of applications.
A. Filter samples to remove particulate and make sure the pH of the mobile phase is within specifications for the column. If the column will be stored for an extended period of time, flush with methanol or acetonitrile.