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Biochemistry Lab Western blot Part 2 – Developing the blot General Instructions Background Immunoblotting (western blots) uses specific interactions of antibodies to detect a protein of interest. Western blotting can be divided into two steps: transfer of the protein from the gel to the matrix (paper) and detection of the epitope with antibodies. Last class we transblotted the SDS-PAGE gel onto the PVDF membrane (transfer paper) and left the blot in blocking solution. We will now finish the second half of the immunoblot, the detection. color. The product of the alkaline phosphatase with the BCP/NBD is insoluble and will permanently precipitate onto the paper. This type of detection gives very sharp bands with low background staining of the membrane. This is one of the easiest methods for western blot detection. There are two disadvantages to this method: the signal or bands fade over time when exposed to light, and second, it is not as sensitive as the some of the other methods such as chemiluminesence. There are several methods to develop a western blot including radioactivity, The steps are listed below. Blocking chemiluminescence, with dry milk covers un-reacted fluorescence and colormetric portions of the blotted paper. systems. Each of these Remember the only protein on the paper methods uses a series of is from the gel. If we were to add the incubations and washes to antibodies directly to the paper most of allow specific antibodies to the primary antibody will non-specifically react with the antigens or bind to the paper. The blot is then target proteins. The antibody washed off with salt and detergent that recognizes the protein on (tween 20) which is essential the paper is in washing to eliminate considered the Enzymatic detection of membrane-‐bound antigens. overall non-specific primary antibody. A 1. Dry milk blocks the unoccupied sites on the hydrophobic interactions. At second antibody, membrane. 2. Primary antibody to a specific antigen is 0.5%, tween 20 will not incubated with the membrane. 3. An antibody enzyme known as the disrupt binding of primary conjugate is added to bind to the primary antibody. 4. secondary antibody, Color d evelopment r eagent i s t hen a dded t o t he b lot. antibodies to antigens, but is then used to detect The HRP or AP enzyme catalyzes the formation of a will optimize detection by the primary. This colored precipitate from the substrate at the site of the eliminating the non-specific secondary antibody is antigen-‐antibody complex. interactions. Inclusion of the often linked to an blotting solution will help reduce non-specific enzyme that will allow for the detection of a interactions with proteins from the gel and the protein. Chemiluminecent techniques use antibody (nothing works perfectly). The secondary antibodies conjugated (linked) to primaries are washed away and secondary horseradish peroxidase to generate light antibody added. Again the blotting buffer is wherever the complex of antigen, primary and included to reduce background and nonsecondary proteins/antibodies are located. specific signals. The secondary antibodies are This signal is detected by exposing the blot to washed away and color substrate is added to x-ray film. develop the blot. We will be using secondary antibodies TTBS / PBST – Tris/PBS buffered saline conjugated to alkaline phosphatase. This solution with Tween 20: A buffered (Tris or enzyme will metabolize the substrate PBS) solution with salt and detergent to limit (bromochloroindoyl phosphate/nitro blue weak, non-specific protein interactions. tetrazolium, or BCIP/NBT) to give a blue-black Biochemistry Lab Western blot – Part 2 Western Blot Procedure: Buffers: PBST or TTBS: as needed for washing. Blocking Buffer: 20 ml of blocking buffer (see last expt for description) to use in 1o and 2o antibody. 1. Wash the blot. • Pour out old blocking solution and rinse with 5-10 ml of TTBS/PBST. • Incubate at room temp with shaking for 2 min. 2. Incubate with primary antibody. • Decant TTBS/PBST • Add in 5 ml of 1:1000 primary antibody in diluted blocking buffer • Incubate at room temp with rocking for 45-60 min. 3. Wash the blot 3 times. • Pour out old solution and rinse with 10-20 ml of TTBS/PBST. • Incubate at room temp with shaking for 3-5 min. • Repeat last two steps a total of three times. 4. Incubate with secondary antibody. • Pour out TTBS. • Add in 10 ml of 1:10,000 secondary antibody diluted in blocking buffer. • Incubate at room temp with shaking for 30 min. 5. Wash the blot 3 times. • Pour out old solution and rinse with 20 ml of TTBS/PBST. • Incubate at room temp with shaking for 5 min. • Repeat last two steps a total of three times. 6. Develop Blot (do this step just before needed). • Pour out old solution. • Add 100 µl of BCIP and 100 µl NBT to 10 ml of color developing buffer (Note: the stock color developing buffer is 25X). • Incubate with shaking at room temp until color develops. 7. Wash with water and let dry on test tube rack in drawer. 8. Take picture of blot results with camera. Biochemistry Lab Western blot – Part 2 SDS PAGE – Western Blot Homework (25 pts): Answer each of the following bullets for the homework assignment. • • • Following the gel electrophoresis and coomassie blue staining, take a picture of the gel. Also take an image of your Western blot. Create publication quality figures of both the gel and the blot WITH figure legends (two separate figures). Label both the SDS PAGE and Western blot as shown in the SDS PAGE instructions from part one. If you are unsure on how to create a publication quality image, please refer to the lab notebook/lab math handout AND/OR use an example from the Journal of Biological Chemistry (www.JBC.org). Measure the distance of migration of the proteins as well as that of the tracking dye (bromophenol blue). Distance of migration is measured from the beginning of the resolving gel to the center mass of a protein band. o Calculate the Rf values for each of the protein standards and the purified protein using the formula below: Rf = Distance of protein migration / distance of tracking dye migration (edge of gel) o o Plot the log of the known protein molecular weights as a function of the Rf. The area in the middle of the gel should yield a straight line. Interpolate the molecular weight of the protein from the graph IMPORTANT POINT – CRITICAL THINKING! How do you know which band on the gel is your protein? How can you identify the protein as your purified protein? Just having a band or the biggest, thickest SDS-PAGE band doesn’t mean it is the protein you are interested in. Does your Rf calculated molecular weight match that which you calculated from the amino acid sequence? How does this relate to the Western blot? • Compare the gel vs. the blot. Use the following points to write up a description of your results: Does the band you believe is your pure protein stained in the SDS-PAGE gel correlate to the band identified by the Western blot? Based on the gel, how pure is the preparation? Are there other protein bands that are found in the Western blot? If there are, why are those bands in the blot present? How does the molecular weight of your purified protein, as determined by the SDS-PAGE gel, match the results determined by the amino acid sequence? If the two values are different, why? Don't assume the differences are mistakes or one is better than another. Instead think of the different conditions that each measurement was taken under and relate that to the differences in results.