Readouts: DNA sequence, Protein abundance
Antibodies are useful tools in the lab. Today you will use antibodies to detect a protein on a blot. This technique, called Western analysis, can give you information about the size and concentration of the protein in the pool that was separated by SDS-PAGE. In your case today, you will use a Western to identify the expression of the light sensing protein, Cph8, and the mutant versions of it. In general, detection depends on which antibody you choose, and the quality of your results depends largely on the quality of that antibody.
For Western analysis, a high quality antibody can have a relatively low affinity for its target protein. This is because the target is localized and concentrated on a blot, allowing the antibody to bind using both antibody “arms” thereby strengthening the association. Even an antibody that is loosely bound to the blot under these circumstances may dissociate then re-associate quickly since the local concentration of the target protein is high. The lower limit for protein detection is approximately 1 ng/lane, a value that varies with the size of the protein to be detected and the Western blotting apparatus that is used. For most acrylamide gels, the protein capacity for each lane is usually 100 to 200 ug (that would be 20 ul of a 5-10 ug/ul protein preparation). Thus 1 ng represents a protein that is approximately 0.001-0.002% of the total cellular protein (1 ng out of 100,000-200,000 ng). Obviously proteins that make up a more significant fraction of the total protein population will be easier to detect.
Many species can be used to raise antibodies. Most commonly mice, rabbits, and goats are immunized, but other animals like sheep, chickens, rats and even humans can be used. The protein used to raise an antibody is called the antigen and the portion of the antigen that is recognized by an antibody is called the epitope. Each antibody can recognize only a small portion of its antigen, typically 5 to 6 amino acids. Some antibodies are monoclonal, or more appropriately “monospecific,” and recognize one epitope, while other antibodies, called polyclonal antibodies, are in fact antibody pools that recognize multiple epitopes. We will be using a monoclonal antibody against the HA epitope today, but for the sake of completion, the origin of both polyclonal and monoclonal antibodies are described.
To raise polyclonal antibodies, the antigen of interest is first purified and then injected into an animal. To elicit and enhance the animal’s immunogenic response, the antigen is often injected multiple times over several weeks in the presence of an immune-boosting compound called adjuvant. After some time, usually 4 to 8 weeks, samples of the animal’s blood are collected and the cellular fraction is removed by centrifugation. What is left, called the serum, can then be tested in the lab for the presence of specific antibodies. Even the very best antisera have no more than 10% of their antibodies directed against a particular antigen. The quality of any antiserum is judged by its purity (that it has few other antibodies), its specificity (that it recognizes the antigen and not other spurious proteins) and its concentration (sometimes called its titer). Animals with strong responses to an antigen can be boosted with the antigen and then bled many times, so large volumes of antisera can be produced. However animals have limited life-spans and even the largest volumes of antiserum will eventually run out, requiring a new animal for immunization. The purity, specificity and titer of the new antiserum will likely differ from that of the first batch. High titer antisera against bacterial and viral proteins can be particularly precious since these antibodies are difficult to raise; most animals have seen these immunogens before and therefore don’t mount a major immune response when immunized. Antibodies against toxic proteins are also challenging to produce if they make the animals sick.
Monoclonal antibodies overcome many limitations of polyclonal pools in that they are specific to a particular epitope and can be produced in unlimited quantities. However, more time is required to establish these antibody-producing cells, called hybridomas, and it is a more expensive endeavor. Antibody-secreting cells are first isolated from an immunized animal, usually a mouse, and then fused with an immortalized cell line such as a myeloma. The fusion can be accomplished by incubating the cells with polyethylene glycol (antifreeze), which facilitates the joining of the plasma membranes of the two cell types. A fused cell with two nuclei can be resolved into a stable hybridoma after mitosis. The unfused antibody-secreting cells have a limited lifespan and so die out of the hybridoma population, but the myelomas must be removed with some selection against the unfused cells. Production of stable hybridomas is tedious and difficult but often worth the effort since monoclonal antibodies can recognize covalently-modified epitopes specifically. These are invaluable for experimentally distinguishing the phosphorylated or glycosylated forms of an antigen from the unmodified forms.
Making antibodies is big business since they can be useful therapeutics. The 2002 market for monoclonal therapeutic antibodies was estimated at almost $300 million and total therapeutic antibody market was estimated at more than $5 billion. These markets are expected to grow considerably, although successful antibody treatments may require clever engineering discoveries to “humanize” antibodies raised in other animals, as well as speedier development, well-protected patents, improvements in drug-delivery methods and cost efficient production of the therapeutics.
Part 1: Probe Western blot
- You should retrieve the blot that you made last time and pour the TBS-T + milk solution into a 50 ml conical tube. Rinse the blot once with TBS-T (volume isn't critical here...you're just trying to rinse the milk out of the container).
- Wear gloves and cut the blot next to the markers in the middle of the blot (if it hasn't been cut last time).
- Place the blot lanes 1-5 in one blotting container, and the other portion of the blot (lanes 6-10) in another container.
- In a conical tube add 10 ul of anti-HA antibody to 10 ml of TBS-T (No milk).
- Add diluted primary antibody to your blot.
- Cover the containers, label with your team color, and place on the platform shaker that's in the chemical hood for 45 minutes. During this time you can set up the β-gal assays of your light and dark grown cells (Part 2, below).
- Pour the antibody solution into a conical tube, writing the identity of the antibody and the date on the tube.
- Give the blots a quick rinse with TBS-T, enough to cover the blot (volume is not critical here).
- Wash the blot on the platform shaker 2 times with TBS-T at room temperature, five minutes per wash. Again the volume of the wash solution is not critical.
- Add secondary antibody (1:1000 Goat-antimouse-alkaline phosphatase) in 15 ml TBS-T and incubate on the platform shaker at room temperature for 30 minutes. During this time you can analyze your sequence data (see Part 3).
- Wash the blot as before (rinse and two washes).
- When you are done washing, mix 250 ul of each of the solutions from the alkaline phosphatase substrate kit into the provided tube of 25 ml 1X developing solution.
- Add developing solution and shake on the platform shaker watching for color to develop. Rinse the blot with water when bands are evident. Recall you anticipated what size protein you are expecting for Cph8. The blots can be left overnight in developing solution or water. One of the teaching faculty will then scan the blot and post the results for you.
Part 2: β-galactosidase Assay
Review protocol presented earlier in this module.
Part 3: 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 (Wed/Fri section: use "astachow" and "be109" instead). 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.
For next time
- Your research article describing this work is due in just over one week. This assignment is due by 11:00 a.m. on the day you have lab. Please turn in your research article electronically by uploading it to the Stellar website that is associated with our class. It is important that you name your file according to this convention: Firstinitial_Lastname_LabSection_assignment.doc, for example: B_Obama_TR_ResArt.doc There will be a 1/3 letter grade penalty for each day (24 hour period) late. If you are submitting your assignment after the due date, it must be emailed to nlerner, lsutliff, nkuldell and astachow AT mit DOT edu.
- Some of you will also be busy preparing a Journal Club presentation for next time. The slides for your presentation should be uploaded to the Stellar website that is associated with our class. The presentation order will be determined by the order that your finished slides are uploaded.
- TBS-T Tris-Buffered Saline + Tween
- monoclonal anti-HA from Abcam, raised in mouse cells
- polyclonal antimouse-AP from BioRad, raised in goat
- BioRad AP detection reagents
- 1 ml 25x detection stock + 24 ml H2O with 0.25 ml solnA and 0.25 ml solnB.