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Protein sample preparation is the first step in western blotting and a key step in determining the success of an experiment. After protein extraction and quantification, protein loading buffers are added to allow protein samples to be separated in subsequent electrophoresis.
Current protein loading buffers are primarily reducing (denatured) protein loading buffers and non-reducing (non-denaturing) protein loading buffers.
A typical antibody can only recognize a linear sequence structure (primary structure) in an antigenic protein. Therefore, in order for an antibody to bind to the epitope, the protein sample needs to be denatured to open its folded spatial structure (except for the antibody specification). In addition to labeling, in general, denatured protein samples are used). The reduced (denatured) protein loading buffer contains the necessary reagents to completely eliminate the high-level structure of the protein. The SDS contained in the buffer can break the intramolecular and intermolecular hydrogen bonds of the protein, causing the molecule to unfold and destroy the secondary and tertiary structure of the protein molecule, while the SDS also binds to the hydrophobic region of the protein due to the large negative charge of the SDS. Therefore, the difference in the original charge between different kinds of proteins is eliminated or masked, so that they all have the same density of negative charges. The strong reducing agent in the buffer, DTT, breaks the disulfide bond between the cysteine residues and the molecules are depolymerized into the polypeptide chain that makes up them. The buffer also contains glycerin to ensure that the protein sample is deposited in the spotted well after loading. The phenol blue dye in the bromine buffer is blue, and its molecular weight is smaller than most proteins. It moves faster than other proteins in electrophoresis, and can monitor the progress of electrophoresis. The protein sample added to the reduced (denatured) protein loading buffer was finally denatured in a 100 ° C water bath for 5 minutes.
The epitope recognized by some antibodies is a three-dimensional structure of a protein composed of non-contiguous amino acids. In this case, a non-denatured protein sample is required for electrophoresis, and the specification of the antibody is generally labeled. Non-reducing (non-denaturing) protein loading buffers do not require SDS and DTT, and protein samples do not need to be denatured.
