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antigen retrieval immunohistochemistry, formalin fixed paraffin embedded, immunohistochemistry protocol, antigen recovery, antigen retrieval buffer, tissue sections, antigen unmasking, antigen retrieval protocol, antigen retrieval pressure cooker, antigen retrieval microwave, antigen retrieval methods, epitope retrieval, antibody, tissue section, immunohistochemistry, paraffin, formalin, citrate buffer, pathology, ihc
A reliable, time tested solution to unmask antigen on formalin-fixed sections
Aptum Biologics Ltd.
38 Baker's Drove
Southampton
SO16 8AD
United Kingdom
W is for Western (blot)
On the basis of the same technology, as our buffers for IHC applications, we offer specially developed buffers for improving sensitivity and decreasing background in Western-blot applications.
The key for every excellent immunoblot result is the signal to noise ratio that your detection system provides. There are two most critical sources of "noise" that decrease the quality and sensitivity or your immunoblot: membrane's free biding spots and non-specific binding of antibodies (the one directed against the detected antigen, and the one from your IgG detection system).
A properly blocked membrane and minimization of the low affinity reactivities of antibody(-ies) allows you to substantially increase the exposure time of your ECL or fluorescent blot to achive a strong specific signal withour risk of proportionally growing intensity of the membrane signal and of the signal from non-specific bands (see the images presented below).
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Western blot was stained with the same antibody using standard blocking procedure (skimmed milk, above) and Western block (bleow). Note the reduction of the noise produced by the blotting membrane.
Effect of antibody diluent on detection of antigen on solid phase with 2 step detection. The primary antibody was diluted in PBS/Tween or in Antibody Diluent W. Signal to noise ratio was 3.42 without and 17.3 with antibody diluent. Note that the specific signal (dots) is equal on both pictures.
Skimmed
Milk
Western
Block
Description & Properties
Western Block, ready-to-use
# AP0500
Specialised solution for blocking free binding sites in Western blots on PVDF, or various types of nitrocellulose membrane. Does not contain large proteins, including serum proteins, BSA, cow milk proteins; low-molecular weight blocking components of this blocking solution do not cause non-specific binding of antibodies to blocking ingredients, while effectively prevent non-specific interactions of test antibody with membrane and transferred proteins on the blot. They also do not prevent (sterically) recognition of the target proteins on the blot due to the size. The buffer is an excellent alternative to self-made skim milk solutions, more effective and improves the signal to noise ratio. Contains ProClin® 300 as preservative and has shelf life of over 12 months at 2-8oC.
Antibody Diluent W, ready-to-use
# AP0501
Specifically designed for Western blot and other solid phase immunoassay, such as immune-PCR, dot blot, protein arrays, this buffer prevents non-specific binding of antibody to solid phase and proteins. As a result, using Antibody diluent may dramatically improve the signal/noise ratio and increase the sensitivity of your assay. In our tests we have seen up to 5 times improved sensitivity due to lower background staining. Contains ProClin® 300 as preservative and has shelf life of over 12 months at 2-8oC.
Stripping Buffer W, ready-to-use
# AP0503
Specially designed for re-probing Western blots, detected with chemiluminiscent and other methods with non-precipitating substrate (i.e. immunofluorescent detection).This formulation effectively and gently removes from blot primary and secondary antibodies, thus allowing to re-probe it again with a new specific antibody.
Washing Buffer W Tris-Based, 10x stock
# AP0502
A concentrated stock for buffer to rinse and wash the western blot between the procedures without removing blocking and other agents preventing the non-specific reactivity of immunochemicals during the detection procedure. Contains ProClin® 300 as preservative and has shelf life of over 12 months at 2-8oC.