![]() Therefore, this technique calls for bright and stable near infrared dyes, such as Alexa Fluor ® 680 and Alexa Fluor ® 790.Western blotting is a commonly used technique in biological research. 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.įluorescent 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. The secondary antibody is incubated on the sample in the same way as usual.Īlexa Fluor® labeled loading controls for fluorescent western blot This indicates if any non-specific binding or false positives may be due to non-specific binding of the secondary antibody.Īntibody dilution buffer containing no antibody is used in place of the primary antibody solution at this point in the procedure. This is when the primary antibody is not added to one strip of membrane. An endogenous positive control is important to validate the results, as well as to indicate how well the reagents (e.g. Most importantly, always ensure the recombinant protein includes the immunogen sequence for the antibody you are using. This is particularly the case with tagged proteins.Īlways ensure tags are placed on the N or C terminal end of the recombinant protein. Folding of the recombinant protein may be different from the endogenous native form, and may prevent access of the antibody to the epitope. There are inherent difficulties with antibody detection of recombinant proteins that need to be considered. This should be an essential part of the experimental plans. We recommend including an endogenous control if you are testing a sample of recombinant protein. View loading controls and loading control guide To check that there has been even transfer from the gel to the membrane during the western blot procedure. To measure the amount of protein present in samples.This gives the researcher confidence that differences in the protein of interest are not due to errors while loading the gel. To check that all lanes in the gel contain the same amount of sample.Loading controls are antibodies to housekeeping proteins, or proteins that are expressed at equivalent levels in almost all tissues and cells. This is to check for non-specific binding and false positive results. If you still have difficulty finding a suitable control, we recommend doing a quick literature search on PubMed to see which tissues and cells express the protein of interest.Ī lysate from a cell line or tissue sample known not to express the protein you are detecting.This will usually provide you with relative levels of expression in various tissues. Check the GeneCards entry for the protein.These can also be considered suitable positive controls. These databases will often have a list of tissues that the protein is expressed in. Try looking at the Swiss-Prot or Omnigene database links on the datasheet.Any tissues, cells or lysates that have been used successfully by these customers can be considered a suitable positive control. Check to see if there are any Abreviews for the antibody. ![]() Not all the datasheets will have a suggested suitable control, and we recommend the following in these circumstances: Always ensure the tissue or cell line you use is from a tested species. We recommend you check the antibody datasheet, which will often provide a suggested positive control. It will verify that any negative results are valid. A positive result from the positive control, even if the samples are negative, will indicate the procedure is optimized and working. A positive control lysate is a lysate from a cell line or tissue sample known to express the protein you are detecting.
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