We have collected a set of frequently asked questions about TidyBlot Western Blot Detection Reagent:
TidyBlot Western Blot detection reagent: Frequently Asked Questions
TidyBlot Reagent (STAR209P and STAR209PA) is a proprietary HRP conjugated secondary detection reagent, which only detects native IgG antibodies. TidyBlot is not an antibody but can be used like a secondary antibody in western blot experiments.
TidyBlot has three advantages compared to conventional HRP conjugated (heavy and light chain) secondary antibodies
TidyBlot is a substitute for a HRP conjugated secondary antibody and is therefore used in the same manner. The recommended TidyBlot dilution range is 1/40 to 1/400 (depending on the abundance of the protein of interest and the substrate used). In our experience a 1/200 dilution of TidyBlot in 5% non-fat milk yields more than 20 (STAR209P) or 40 (STAR209PA) mini western blots when using Clarity™ Western ECL Blotting Substrate.
TidyBlot preferentially binds to the native antibody used during the western blot procedure rather than the SDS-denatured/reduced form present in lysates/immunoprecipitates. Best results are therefore achieved by fully denaturing the immunoprecipitate/lysate (consider the duration of sample boiling to reduce IgG disulphide bonds and the type/concentration of reducing agent). TidyBlot is used in the same manner as conventional HRP conjugated secondary antibodies.
TidyBlot is compatible with the below listed IgG monoclonal and polyclonal antibodies:
|Human||IgG1, IgG2, IgG4||Compatible|
IgG2a, IgG2b, IgG3
IgG1: affinity for IgG1 varies and may not be strong
Affinity should therefore be tested on an antibody by antibody basis by performing dot blot analysis
TidyBlot is not known to detect IgA, IgD, IgE and IgM antibodies.
If using a mouse IgG1 antibody, perform dot blot analysis to determine compatibility. The TidyBlot Reagent might not detect mouse IgG1. In that case you could try our HRP conjugated Rat Anti-Mouse Kappa Light Chain Specific Antibody, clone OX-20 (MCA152P). This antibody does not detect mouse IgG heavy chains and thereby allows you to detect immunoprecipitated proteins of ~50 kDa without obstruction by heavy chains.
One vial of TidyBlot enables you to perform at least 20 (0.5 ml, STAR209P) or 40 (1 ml, STAR209PA) mini western blots; assuming a 1/200 dilution in 5% non-fat milk using Clarity™ Western ECL Blotting Substrate. As is necessary, for every secondary detection reagent, you will need to establish the optimal dilution by performing a dilution series.
TidyBlot is currently only available conjugated to HRP. Unfortunately, we do not offer TidyBlot in other formats.
Yes, TidyBlot can be purchased in bulk as a multiple of 0.5 ml (STAR209P) and 1 ml (STAR209PA). Please contact your local sales representative for a bulk quote and delivery time.
TidyBlot western blot data have been generated using the Bio-Rad ChemiDoc™ MP System rather than X-ray film. The system has various filters for the detection of fluorophores and other detection reagents based on their excitation and emission wavelengths. Each reagent is assigned a color (red, green or blue) when visualizing the blot with the ChemiDoc Imaging Software. In the case of the TidyBlot data, the chemi channel was used to detect HRP chemiluminescence and assigned the color red. Protein standards (Precision Plus Protein™ All Blue Protein Standards) were detected in the DyLight 650 channel and assigned the color green*.
*Please note that these are pseudocolors and are not a reflection of emission colors.
Bio-Rad offers a HRP conjugated Rat Anti-Mouse Kappa Light Chain Specific Antibody, clone OX-20 (MCA152P). This antibody does not detect mouse IgG heavy chains and thereby allows you to detect immunoprecipitated proteins of ~50 kDa without obstruction by heavy chains. We also offer a comparable product for rabbit; Mouse Anti-Rabbit Light Chain Specific Antibody, clone SB62a (MCA6003P), which is a HRP conjugated secondary antibody that does not bind to rabbit IgG heavy chains.