Pooled AFC (if you have or can “borrow” some)Ĭoncentrated AFC (if you have or can “borrow” some) Table for sample loading and gel lane map* Laneīenchmark Pre-stained Protein Ladder Staff prepares SAVE everything that is left over, after you take your samples. Put all the samples in your freezer box for next week.Aliquot equal parts sample and SDS sample buffer to each tube you make. Use your freezer box samples and the IMAC samples from lab, or get sample from another group if necessary.The lab manager prepares the highlighted tubes, you don’t need to make those. Label tubes first, clearly by lane number! The table amounts will make enough for the 2 gels next week.Note that you are making more than you will actually load next week. Prepare ONE SET of tubes listed below (Samples 3 – 15). To test our own samples (both Aim1 and Aim2 on one gel), we will load controls, beef heart samples and a few IMAC samples for analysis (and to compare to the gel provided). Decide which lab partner will load these samples into 2 gels: one to be stained and one transferred.īelow in blue text is the loading of a gel found in Aim1 for a gel that will be provided. Load your samples in the order as listed below. If you used the Duo-Flow protocol, share samples with a group that used the Manual, Qiagen Kit protocol and vice versa. Load Everything in the tubes we give you.ĭo not put the samples into new tubes, simply boil them Except for the M3913 Ladder ( and do Not boil the Benchmark ladder ) We Provide Samples For Lanes 2 – 6 for both Protocol 1 and Protocol 2. Procedure 1 SDS-PAGE gels of Expressed Protein Purification Here is the loading and protocol for the Expressed Protein (as if we were running all the samples we expressed and purified from our clones). Be sure to look at how the lanes are loaded.You will also load a few IMAC samples to compare to this.We are providing a gel with all the samples run for you to use as a reference.You have purified protein using one method (Qiagen IMAC) and have the methods for the other method (FPLC IMAC).You will predict what you expect to see detected by anti-LDH antibodies. No Pre-Lab questions for lab 11, but still write out an abbreviated protocol and a summary of last week’s lab. Again, this involves making a set of known molecular weight standards and extrapolating. Molecular Weight Determination – Similar to size exclusion chromatography, one can plot the log of the MW vs the relative mobility (Rm) of the protein and determine molecular weight of an unknown protein. Note: you can even stain a Western blot with Ponceau S to see bands and then rinse. More sensitive stains have been developed, like silver stain and Ponceau S. After staining, excess dye is washed away and the tightly bound dye is visualized (right). After the gel is run, it needs to be stained (and fixed) with Coomassie Brilliant Blue. Staining the gel – There is a tracking dye (usually bromophenol blue) that is added to your sample so that you can follow the running of the protein. If proteins of different sizes are uniformly decorated they will separate just based on size (Stoke’s radius) regardless of differences in native protein surface charge. The detergent binds to the protein via its hydrophobic region and presents the minus charge to the solution (illustrated below). SDS-PAGE electrophoresis – Sodium dodecyl sulfate is strong anionic detergent that will decorate proteins and give them an overall negative charge. Think about the process from lysis to imaging. INQUIRY: How would you plan an experiment using gel-electrophoresis to visually see purification and to visualize a protein dimer versus a monomer?
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |