Searching for Proteins: Western Blots
My lab is interested in a specific protein called Caveolin and as a result there are a variety of techniques they use to quantify protein expression level. One of these methods is a Western Blot.
Western Blots:
Western Blots are a technique used to probe for the expression of a specific protein. This is done by taking a homogenized protein mixture from cells or tissues and running them through a gel. The gel has electricity running through it and this separates proteins by weight. Then the proteins on the gel are transferred to a membrane. Then the membrane is socked in a primary antibody that specifically attaches to a protein. Then, after being rinsed with the primary antibody the membrane is then soaked in a solution containing a nonspecific antibody that binds to the primary antibody. The secondary antibody contains a fluorescent tag that allows it to be seen using a special infrared machine. Depending on the location and width of the fluorescent bands, one can compare the difference in protein expression between two groups. Below are the major steps for running a Western Blot.
1. Bradford: The purpose of a Bradford is to find the concentration of protein in the different samples. If the samples all had a different concentration of protein, it would be flawed to think that the bigger band of protein on the membrane is due to a difference in the samples. This is done by first making standards with known concentrations of proteins. Then a dye is added which changes color when it attaches to the protein. The wavelength of the color is linearly correlated with the amount of protein present. Then the dye is also put in the samples and their wavelength is noted. Using the equation obtained from standards, the amount of protein can be calculated. Then, the mL of sample needed for the ideal amount of protein for the Western Blot can be obtained.
2. Loading the Gels: After calculating the amount of protein needed and the dilutions, the samples are then mixed with loading buffer. Loading buffer contains heavy molecules such as glycerol which makes the mixture heavier and thus more likely to sit at the bottom of the lanes of the gel. Then, the samples are heat shocked for about ten minutes and left on ice for another ten minutes. Then ten microliters of each sample is loaded onto each lane. The ladder is also added to the gel. The ladder contains fluorescent molecules that will later serve as reference weight point that other proteins can be compared to. After adding the samples to the wells, the gels are placed in containers suspended in running buffer. Then an electric current will be passed through them for an hour or two. This electric current prompts the proteins to move through the gel. The lighter proteins will need less time to travel through the gel as they are less likely to be caught in the pores. Therefore, the proteins at the lower end of the gel have a smaller weight than the proteins near the top.
3. Transfer: The proteins on the gel are then transferred onto a membrane. The membrane and the gel are closely packed together and suspended in transfer buffer. Then, electricity flows through and transfers the proteins onto the membrane.
4. Blocking: The membrane then sits in a bovine serum albumin solution (BSA). Since the membrane has a high affinity for proteins, the BSA attaches to membrane and prevents antibodies from binding to the membrane.
5. Adding the antibodies: First the primary antibody is added to the membrane. The primary antibody specifically binds to the protein of interest. Then, a secondary antibody is added. This antibody is general and will bind to any antibody of a specific animal. The secondary antibody has a fluorescent tag that can be detected.
6. The width of the florescent band represents the amount of protein there is. This method is useful if one is trying to see the difference in protein expression between two different groups.
