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A positive control lysate is a lysate from a cell line or tissue sample known to express the protein you are detecting. A positive result from the positive control, even if the samples are negative, will indicate the procedure is optimized and working. It will verify that any negative results are valid.
We recommend you check the antibody datasheet, which will often provide a suggested positive control. Always ensure the tissue or cell line you use is from a tested species. Not all the datasheets will have a suggested suitable control, and we recommend the following in these circumstances:
A lysate from a cell line or tissue sample known not to express the protein you are detecting. This is to check for non-specific binding and false positive results.
Loading controls are antibodies to housekeeping proteins, or proteins that are expressed at equivalent levels in almost all tissues and cells.
Loading controls should be used:
We recommend including an endogenous control if you are testing a sample of recombinant protein. This should be an essential part of the experimental plans.
There are inherent difficulties with antibody detection of recombinant proteins that need to be considered. Folding of the recombinant protein may be different from the endogenous native form, and may prevent access of the antibody to the epitope. This is particularly the case with tagged proteins.
Always ensure tags are placed on the N or C terminal end of the recombinant protein. Most importantly, always ensure the recombinant protein includes the immunogen sequence for the antibody you are using. An endogenous positive control is important to validate the results, as well as to indicate how well the reagents (e.g. antibodies) and procedure are working.
This is when the primary antibody is not added to one strip of membrane. Secondary antibody only is added. This indicates if any non-specific binding or false positives may be due to non-specific binding of the secondary antibody.
Antibody dilution buffer containing no antibody is used in place of the primary antibody solution at this point in the procedure. The secondary antibody is incubated on the sample in the same way as usual.
With the advent of fluorescent western blotting, multiple proteins can now be analyzed simultaneously using different fluorophores.This growing technique has been proven to provide improved linearity and increased reproducibility when compared to standard chemiluminescent detection methods in Western blot.
Fluorescent western blotting works optimally in the near-infrared region of the spectrum in order to avoid the chance of membrane autofluorescence within the visible light range.
Therefore, this technique calls for bright and stable near infrared dyes, such as Alexa Fluor® 680 and Alexa Fluor® 790.
|Target||Alexa Fluor® 680||Alexa Fluor® 790|
Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.