The mutant phenotypes you're following might be explained by changes in kinasing activity of Cph8. They could also be explained by changes in protein expression. For example, if the protein's concentration is 10X that of wild type, then it might increase the pool of kinased OmpR, resulting in greater LacZ activity when the cells are grown in the dark even though this would have ~ nothing to do with the kinasing reaction rates. Thus it's important to show that the protein still exists in the cell (at a minimum) and that its expression is about the same as the wild type protein (ideally). With a Western analysis, you will compare the expression levels of the wild type and mutant proteins, detecting the various Cph8 versions using an antibody that was raised to a His6-tagged version of EnvZ.
There are some important differences in protein and DNA gel electrophoresis. One thing you’ll notice right away is that the gel itself is different. DNA molecules are typically separated thorough an agarose matrix whereas acrylamide is used for proteins. Both are porous sieves that retard molecules based on their length, with smaller molecules moving through the matrix faster than longer molecules. Agarose gels are run horizontally and acrylamide gels are set in the tank vertically but gravity has nothing to do with either separation. Electrical poles draw the charged molecules through the matrix. Unlike DNA, proteins do not have a uniform charge so before electrophoresis they are coated with a charged molecule (called SDS) to add negative charge proportional to their length. You could expect proteins of identical length but folded into different shapes to separate differently (not the desired outcome) so proteins are also unfolded before they are loaded on a protein gel. This is done by boiling them in the presence of a reducing agent, breaking disulfide bridges and denaturing the protein. The last notable difference in DNA and protein electrophoresis is the visualization techniques used to find the molecules once they’ve passed through the gel. Recall that DNA was visualized with Ethidium Bromide, an intercalating dye that changes its fluorescence when bound to DNA. Proteins can be detected with Coomassie stain, which detects abundant proteins in the gel, turning them blue. Other more sensitive techniques are available for staining protein gels (for example silver stain). Finally, a Western blot can be used, as you will today, to transfer the proteins from the gel to a membrane that is later probed with an antibody specific to the protein of interest. In our case we will use a polyclonal antibody to detect the EnvZ portion of Cph8.
Today is a busy day! In addition to running your protein gel and blotting the proteins to a nitrocellulose membrane, you will also look at the sequencing data if it is available as well as prepare bacterial photographs with your new strains. Finally, if you choose, you can measure once the β-gal activity associated with your samples one more time, looking in particular at the units when cells were grown in the light and the dark.
Part 1: Protein Gel (SDS-PAGE)
Each group will run a lane of molecular weight markers, a lane with a positive control for the Western (e.g. an aliquot of purified His6-EnvZ), a lane with the wild type light sensor, and two lanes mutants from your library screen. Two teams will share one gel.
- Retrieve the bacterial cultures carrying the wild type or mutant light sensors that have been grown for you.
- To compare intensities between lanes on the protein gel it's necessary that equal numbers of cells be loaded into each well. You'll assess the number of cells in each sample by making a 1:10 dilution of the three strains in Z-Buffer and use the spectrophotometer to measure the density of the samples at a wavelength of 600 nm. This measurement tells you something about the number of cells in a millileter of liquid. For example a reading of 0.7 says the sample has 0.7 OD units of cells / ml.
- Calculate the volume of your cells needed to give 4 OD. Thinking again about a sample that reads 0.7 OD: if you wanted to collect the number of cells equivalent to 4 OD unit, then you would have to collect 4/0.7 = 5.7 ml of that sample to get 4 OD's worth of cells. Heads up: don't forget that your spectrophotometric reading is for a 1:10 dilution of the original (undiluted) samples, so if you go back to the overnight cultures you'll have to take that dilution factor into account.
- Move the calculated volume of cells to well-labeled eppendorf tubes, and spin the tubes in a microfuge for 1 minute to pellet the bacteria. Be sure that each eppendorf is balanced in the microfuge with an opposing eppendorf containing the same volume.
- Remove the supernatant and resuspend each pellet in 100 ul of "EasyLyse" protein extraction solution (a commercial product from a company called Epicentre). Incubate the solutions at room temperature for 5 minutes, then pellet the debris by spinning the tubes in the microfuge (full speed) for 2 minutes. The supernatant is the lysate that you will run on your protein gel.
- Mix 30 ul of the bacterial lysates with 30 ul of 2X sample buffer. Sample Buffer contains glycerol to help your samples sink into the wells of the gel, SDS to coat amino acids with negative charge, BME to reduce disulfide bonds, and bromophenol blue to track the migration of the smallest proteins through the gel. Wear gloves when using sample buffer or your hands will get blue and smelly.
- Prepare your positive control H6-EnvZ protein tube by moving 50 ul of the sample to an eppendorf tube.
- Put lid locks on the eppendorf tubes and boil for 5 minutes.
- Put on gloves. Load the indicated volumes of each sample onto your acrylamide gel in the order below. Once you have loaded a sample from one tube, move it to a different row in your eppendorf tube rack. This will help you keep track of which samples you have loaded.
10. Once all the samples are loaded, turn on the power and run the gel at 200 V. The molecular weight standards are pre-stained and will separate as the gel runs. The gel should take approximately one hour to run. During that hour, you should work on part two of today's protocol.
11. Wearing gloves, disassemble the electrophoresis chamber.
12. Blot the gel to nitrocellulose as follows:
- Place the gray side of the transfer cassette in a tupperware container which is half full of transfer buffer. The transfer cassette is color-coded so the gray side should end up facing the cathode (black electrode) and the clear side facing the anode (red).
- Place a ScotchBrite pad on the gray side of the cassette.
- Place 1 piece of filter paper on top of the ScotchBrite pad.
- Place your gel on top of the filter paper.
- Place a piece of nitrocellulose filter on top of the gel. The nitrocellulose filter is white and can be found between the blue protective paper sheets. Wear gloves when handling the nitrocellulose to avoid transferring proteins from your fingers to the filter.
- Gently but thoroughly press out any air bubbles caught between the gel and the nitrocellulose.
- Place another piece of filter paper on top of the nitrocellulose.
- Place a second ScotchBrite pad on top of the filter paper.
- Close the cassette then push the clasp down and slide it along the top to hold it shut.
- Place the transfer cassette into the blotting tank so that the clear side faces the red pole and the gray side faces the black pole.
13. Two blots can be run in each tank. When both are in place, insert the ice compartment into the tank. Fill the tank with buffer. Be sure the stir bar is able to circulate the buffer. Connect the power supply and transfer at 100 V for one hour. You can use this time to complete part 2 of today's protocol.
14. After an hour, turn off the current, disconnect the tank from the power supply and remove the holders. Retrieve the nitrocellulose filter and confirm that the pre-stained markers have transferred from the gel to the blot. Move the blot to blocking buffer (TBS-T +5% milk) and store it in the refrigerator until next time.
Part 2: DNA sequence analysis
If the data from the MIT Biopolymers Facility is available for you to examine, continue with this analysis.
Rather than look through the sequence to magically find the relevant portion, you can align the data with the plasmid sequence for wild type pCph8 from Jeff Tabor and the folks who published the bacterial photography system. Using this approach, the differences will be quickly identified. There are several web-based programs for aligning sequences and still more programs that can be purchased. The steps for using the BLAST web-based tool is sketched here. BLAST is an acronym for Basic Local Alignment Search Tool, and can be accessed for free through the National Center for Biotechnology Information (NCBI) home page
Align DNA with "bl2seq" from NCBI
- Retrieve the sequences from this link. Choose the "Login to dnaLIMS" link and then use "nkuldell" and "20.109" to login. At the bottom of the left panel should be a link to download your sequencing results. Select the appropriate order # (you'll be told which one is correct) and then "submit." From the list find your sample(s). The quickest way to start working with your data is to follow the "view" link. From this link you'll see the sequencing traces and can add the sequence to the workbox by clicking on "sequence text." If there were ambiguous areas of your sequencing results, these will be listed as "N" rather than "A" "T" "G" or "C." It's fine to include Ns in the steps listed below.
- Since the oligo read your sequence in the "reverse" direction, it's recommended that you find the reverse complement of the sequence data. This tool is helpful for finding the reverse complement.
- Paste the reverse complementary sequence into the "Sequence 1" box at the BLAST2 sequences site. The alignment program can be accessed through the NCBI BLAST page or from this link.
- Paste the pCph8 sequence from here into the "Sequence 2" box.
- Align the sequences. Matches will be shown by lines between the aligned sequences. The sequence data from your candidate will be the "query." The sequence data from the original plasmid will be the "subject."
- Print and save a screenshot of the relevant alignment (using shift/command/4 or the Grab program under utilities), and draw conclusions about the alignment in your notebook. You might want to email the alignment screen shot to yourself or post it to your wiki userpage.
Identify Amino Acid changes with Sequence Manipulation Suite
If you've identified a region of the sequence that is not identical in the mutant and the wild type version of the Cph8 protein, then you'll want to know what amino acids that region encodes. You can use the Sequence Manipulation Suite to help you translate the region of interest in all 3 reading frames. The correct translation frame should not have stop codons in the wild type sequence. Again you should print and save a screenshot of the relevant translation, and draw conclusions about the amino acid changes in your notebook. You might want to email the translation screen shot to yourself or post it to your wiki userpage.
Part 3: Bacterial Photograph
Review the protocol presented earlier in this module.
Part 4: OPTIONAL: β-galatosidase Assays
If you set up overnight cultures for your samples in the light and dark to assess the β-gal activity, you can perform those assays today as well. Review the protocol that was presented earlier in this module for details and ask the teaching faculty if you need help.
For Next Time
- To be turned in: You must calculate the expected length of the Cph8 protein using the DNA sequence that's here. If you don't already have a favorite program for doing this calculation, try to use the suite of DNA analysis programs that are here.
- NOT to be turned in: You should update the Materials and Methods section that you are writing for your research article to include the experiments you have performed today.
- Epicentre EasyLyse Solution
- BioRad Ready Gel 4-15% Tris-HCl Gel, cat #161-1158
- SDS-PAGE Loading Dye
- 60 mM Tris, pH 6.8
- 2% SDS
- 1% glycerol
- 0.01% bromphenol blue
- 5% beta-mercaptoethanol
- Running Buffer
- 25 mM Tris
- 192 mM Glycine
- 0.1% SDS
- Transfer Buffer
- 25 mM Tris
- 192 mM glycine
- 20% v/v methanol
- TBS-T Tris-Buffered Saline + Tween + milk
- 20 mM Tris, pH 7.5
- 500 mM NaCl
- 0.1% Tween
- 5% milk
- H6EnvZ + control protein