The first step to creating publication worthy Western blots
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis, phew that was a mouthful! Let’s go with SDS-PAGE for short. This technique is used to separate proteins in a complex sample by size. To keep things simple, I’ll stick to talking about denaturing gels here since they are most commonly used. SDS plays a critical role in breaking down the two- and three-dimensional structures of proteins by adding a negative charge to amino acids. The heating step should always be part of your routine as it breaks down hydrophobic interactions, enabling SDS binding and complete denaturation of proteins. If your protein contains disulfide bonds, add a reducing agent such as DTT, β-mercaptoethanol or TCEP to your sample buffer.
Now that your sample is completely denatured, you will need to decide how much to load onto your gel. If you have a low abundance protein, it may be tempting to load an excessive amount of lysate. In most situations, lysate loads should not exceed 30µg of total protein. Loading more will cause high abundance proteins to deform the lanes of the gel. Also consider that when high abundance proteins are transferred, they may cause saturation due to the limited binding capacity of your membrane. This is especially important to consider when high and low abundance proteins are in the same vicinity. When loading your gel, it is better to load extra lanes of molecular weight marker rather than skipping a lane to avoid distortion. There’s nothing worse than an overloaded lane next door to an empty one.
If you have opted for the convenience of precast gels, make sure to follow the manufacturer’s instructions. Carefully select your running buffer to achieve the desired separation. Many suppliers have tools to aid in choosing the optimal gel percentage and running buffer. Pay close attention to the suggested voltage that may be applied as it is easy for the gel to become overheated and begin to melt. Also, before running a precast gel, don’t forget to remove the sticker located on the bottom of the gel cassette. If you prefer to cast your own gels, check out our gel making tips and tricks post for creating publication worthy gels.
Protein Sample Loading Buffers
Avant Buffer Pouches
FlashBlot Transfer Buffer