Western blotting is a simple and straightforward technique for protein characterization. However, the process entails a great number of workflow steps, and we are committed to providing you with products and guidance that secure you optimal and reliable results. In our Western blot portfolio, you will find a wide selection of products for successful Western blotting; from preparation to separation and blotting as well as antibody incubation and, lastly, detection.
For incubation with antibodies, you need to do blocking, incubate with a primary antibody, wash, incubate with a secondary antibody, and wash a final time. However, direct detection can be suitable in many cases. In our portfolio, you will find a range of products to support antibody incubation; primary antibodies, and blocking buffers.
The preparation of your samples is the first step in Western blotting. It is important to prepare the starting material properly to obtain a successful Western blot. Before sample preparation, you must acquire knowledge about the protein source, protein location, expression levels, controls, hydrophobicity of protein target, and more.
Whether you are working with cultured cell lines or tissue samples, sample preparation is often a process of trial and error in which you, through troubleshooting, need to choose the right lysis buffers, enzyme inhibitors, and appropriate stoichiometry. Some samples may even require mechanical disruption. Let us help you facilitate this process through state-of-the-art products and scientific experience.
Following sample prep, you need to do polyacrylamide gel electrophoresis (PAGE) and blotting. Most often, the samples are treated with the negatively charged SDS to unfold and separate the protein content by size. It is also possible to perform non-reducing gel electrophoresis to maintain the native state of the protein. There are many things you need to consider in the protein separation step; protein concentration and buffer exchange, molecular weight standards, and protein stain, among others.
Blotting is carried out to transfer proteins from the gel to a solid membrane. The blotting is performed by assembling filter papers, a membrane, and a gel into a sandwich. An electrical field allows the proteins from the gel to wander onto the membrane. The effectiveness of the transfer is heavily reliant on the gel acrylamide percentage, the molecular weight of electrophoresed proteins, and the selected blotting membrane. We have sample loading buffers, protein ladders, transfer buffers, and much more for the protein separation and blotting steps of Western blotting.
At the final step of Western blotting, you will need to choose a detection method to visualize your results. We are ready to guide you in the choice between chemiluminescence, fluorescence, and autoradiography for detection. Your choice will influence the protocol and chemistry for your Western blot, depending on the type of information you are looking for.
Our specialists are ready to guide you to successful Western blotting. Take full advantage of our Western blotting experts and let us guide you to the best setup for your project. Our portfolio for Western blotting is full of products from leading manufacturers.
