Basic Experimental Guidelines

Below is a list of basic experimental guidelines for various types of experiments. While this list is not comprehensive (no list could be, as methods and techniques will vary significantly based on the nature of your experiment) we do strongly suggest consulting the guidelines below when planning and conducting your experiment.

ELISA and Biochemical Assay Kits

• ELISA and biochemical assay kits are generally shipped as self-contained units encompassing all of the reagents necessary to carry out the assay. When performing an ELISA assay, ONLY use the reagents that are included with the kit. Do not substitute any component of the kit or use any external reagent unless this substitution is specifically directed or approved by the product manual, the manufacturer, or by antibodies-online.
• Both samples and standards must be run in duplicate at least in order to mitigate the possibility of user/equipment error.
• ALWAYS run the complete complement of standards included in the kit in duplicate each time the assay is performed.
• We always suggest a small pilot experiment using one ELISA kit to ensure the validity of the experimental conditions and the appropriateness of sample dilution (1:1, 1:10, 1:100,...). We recommend to use the standard and a small number of samples as well as negative and positive controls in duplicates.

Western blot

• An appropriate loading control is necessary for every western blot experiment performed. When a secondary antibody is used for labeling, the same secondary should be used to label both experimental and loading-control samples. Antibodies-online offers a wide variety of antibodies that are compatible with commonly used loading controls. For more information, please consult one of our technical support scientists.
• Blocking your membrane prior to staining is necessary to prevent nonspecific binding. Antibodies-online recommends use of a solution containing 5% nonfat dry milk, 2-3% BSA, or any of the common commercially available blocking buffers approved as suitable for Western blot applications.
• If the antibody does not appear to recognize your target, it may be helpful to ensure that the target protein is actually expressed in the tissue or cell line you are investigating. Antibodies-online accepts experimental evidence confirming expression at the proteomic level, as well as canonical evidence (e.g. a reference in a peer-reviewed journal).
• Signal strength in a Western blot experiment is highly dependent upon the concentration of the antigen in the sample tested, and can vary significantly depending upon the primary and secondary antibodies used. It may be necessary to repeat the same experiment with different primary/secondary antibody dilutions in order to achieve the desired result.

IHC/IF

• Many antibodies are only approved and tested on tissue sections that have been preserved via a specific method. For example, antibodies marked IHC(p) or IF(p) are approved only for use on FFPE (formalin fixed and paraffin embedded) tissue sections after antigen retrieval. While this certainly doesn’t preclude an ability to function in non-approved applications, antibodies-online cannot extend a guarantee to an application that hasn’t been specifically approved by the manufacturer.
• When performing IHC/IF on FFPE tissue sections, antigen retrieval may be necessary to unmask an epitope recognized by some antibodies. If you need a specific protocol for antigen retrieval, please contact one of our technical support scientists.
• Blocking your sample is necessary to prevent nonspecific binding and excessive background signal. Antibodies-online recommends use of a solution containing 5% nonfat dry milk, 2-3% BSA, or any of the common commercially available blocking buffers approved as suitable for IHC/IF applications.
• Antibodies-online accepts experimental evidence confirming expression at the proteomic level, as well as canonical evidence (e.g. a reference in a peer-reviewed journal).
• Signal strength in an IHC or IF experiment is highly dependent upon the concentration of the antigen in the sample tested, and can vary significantly depending upon the primary and secondary antibodies used. It may be necessary to repeat the same experiment with different primary/secondary antibody dilutions in order to achieve the desired result.

Flow Cytometry/FACS

• Isotype controls are always strongly recommended when performing FACS/Flow Cytometric analysis. A properly matched isotype control helps to establish background, and allows the user to differentiate between signal and noise. If you need help selecting an appropriate isotype control, please contact one of our technical support scientists.
• Cytosolic and other intracellular antigens will require membrane permeabilization prior to FACS analysis. Permeabilization reagents and protocols will depend upon the cell type, and membrane type that needs to be permeabilized. If you need assistance with your specific pemeabilization protocol please contact one of our technical support scientists.
• Successful FACS/Flow cytometry analyses are highly dependent on the density of cells used. The optimal cell density will vary depending on cell type, antigen(s) measured, and the method of analysis. It may be necessary to try several different cell densities in order to achieve the desired result.

Recombinant Proteins

• It is important to note that, while every effort is made to preserve endogenous structure and function during recombinant protein production, recombinant proteins may not share identical properties with their native counterparts.
• Differences in protein folding and post-translational modifications can result in a recombinant protein that has different properties than its native counterpart. These differences may affect function, and (in some cases) the ability of an antibody to recognize the protein in question.
• Unless specifically noted on the datasheet, or confirmed by our technical support staff, antibodies-online cannot guarantee functional activity of any recombinant protein, nor can we guarantee compatibility between any antibody and corresponding recombinant protein.

If you need any clarification, are looking for appropriate controls, or would like to find basic guidelines for any other type of experiment please contact us. We would be happy to help you plan your next experiment.