Creative Biolabs is one of the well-recognized experts who are professionals in antibodies development and antibody-related services. Our experienced scientific support team now answers your commonly frequently asked technical questions (FAQs).
Secondary antibodies should be raised based upon the primary antibody you are using that produced by different host species. If your primary antibody is a mouse monoclonal, the secondary antibody should be an anti-mouse Ab. Similarly, a rabbit monoclonal primary antibody will correspond to an anti-rabbit secondary Ab. In addition, secondary antibodies conjugated to fluorochromes and chromogens depend on your project requirements.
The optimal antibody concentration must be determined experimentally for each assay, which gives the best results with the minimum background. Usually, an ideal dilution ratio is determined by using a series of dilutions in a titration experiment. For example, if a product datasheet suggests using a 1:200 dilution, it is recommended to make dilutions of 1:50, 1:100, 1:200, 1:400 and 1:500.
In addition, a titration experiment is performed by first selecting a fixed incubation time and then a series of experimental dilutions of the antibody. Please note that each dilution ratio should be tested on the same type of sample in order to keep the same experimental conditions.
The size of the aliquots mainly relies on how much one typically uses in an experiment. We recommend aliquots should be no smaller than 10 µL. The smaller the aliquot, the more the storage concentration is influenced by evaporation and adsorption of the antibody onto the surface of the storage vial.
Isotype controls are a type of negative control designed to confirm that the primary antibody binding is specific. Generally, some non-specific background signal is caused by primary antibodies that can bind to Fc receptors non-specifically present on the cell surface or other protein interactions. Thus, the selection of isotype control antibody to measure the non-specific signal is important, and the isotype control Ab should match the primary antibody’s host species, isotype and conjugation.
To find a suitable isotype control, you can contact us and tell us the species and subclass of your primary antibody. We will provide you a variety of available isotype controls to meet your requirements.
In scientific testing, a positive control group is critical that is a control group not exposed to the experimental treatment but exposed to some other treatment, which is known to produce the expected effect.
To choose a suitable positive control, you can search at the Swiss-Prot or Omnigene database that will often have a list of tissues that the protein is expressed in. Additionally, GeneCards also shows relative expression levels of a protein in various tissues that will help you find a suitable positive control. Lastly, a quick literature search on PubMed to see which tissues and cells express the protein of interest is also an effective method to choose a suitable control.
Freezing at -20°C or -80°C in small aliquots is the most suitable storage condition for most of antibodies, due to aliquotting can minimize damage during freezing and thawing, and contamination introduced by pipetting from a single vial multiple times. Aliquots should be frozen and thawed once and kept at 4°C. In most cases, it is acceptable to store the antibody at 4°C for one to two weeks upon receiving it.
We can provide some of our antibodies cross-reactivity data, please contact us if you need. In addition, you also can confirm the cross-reactivity by checking the sequence alignment of the immunogen with the isoforms or other proteins you are interested in. Usually, if an alignment score is over 85%, which indicates that the antibody may cross-react. Thus, no cross-reactivity requires the percentage alignment to be much lower than 85%.
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