20.109(S14):Begin Western protein analysis (Day2)

Introduction

Topic 1: Reiterate purpose of WB, now some details about cell lines

Topic 2: Basis of WB, including background on antibodies

S13 M3 starter A great way to identify a specific protein from a complex mixture is to exploit antibodies – also called immunoglobulins – whether in a Western blot or by ELISA (enzyme-linked immunosorbent assay). In native physiological settings (such as your own body), antibodies are secreted by B cells in response to pathogens. A given antibody is highly specific ([math]\displaystyle{ K_D }[/math] ~ nM) for its binding partner, called an antigen, and the entire antibody population for a given person is incredibly diverse (>10⁷ unique antibodies). Diversity is maintained by recombination processes at the DNA level, and specificity entailed by protein structure.

Antibody proteins comprise constant (C) and variable (V) regions, on both their heavy and light chains. The C regions determine antibody effector functions, such as antibody-dependent killing of infected cells. The three hypervariable portions of the V region together make up the antigen-recognition site. Only a small portion of an antigen, called an epitope, is recognized by its cognate antibody. This ~10 amino acid region may be linear, or it may be made up of linearly distant regions and thus recognized only when the antigen is in its native conformation.

Antibodies can be raised in animals or special cell lines and even be genetically engineered. Polyclonal antibodies (pools of antibodies that recognize distinct epitopes on the same antigen) can be obtained from animal serum. The animal is infected with the antigen of interest in the presence of a costimulatory signal, usually multiple times, and then bled. In this case, a large fraction of the antibodies obtained will not be against the antigen of interest. In contrast, monoclonal antibodies can be made both highly specific and pure. In this process, normal antibody-producing B cells are fused with immortalized B cells derived from myelomas, and the two cell types are fused by chemical treatment with a limited efficiency. To select only heterogeneously fused cells, the cultures are maintained in medium in which myeloma cells alone cannot survive (often HAT medium). Normal B cells will naturally die out over time with no intervention, so ultimately only the fused cells, called hybridomas, remain. Genetic engineering can be used to combine a human antibody ‘frame’ (all of the C and part of the V region) with an antigen-recognition site discovered in another species (e.g., murine). When antibodies are used as therapeutics, this approach decreases the possibility that the patient’s body will treat them as foreign, compared to an antibody produced from only mouse genes. Normally, injecting an antibody from species X into an animal of species Y will cause production of anti-X antibodies, called secondary antibodies. These can be very useful in technical assays, as you will see below.

F13 M2 starter

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.

generating polyclonal antibodies

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.

generating monoclonal antibodies

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. Whereas the 2002 market for monoclonal therapeutic antibodies was estimated at almost $300 million, sales grew to $43 billion in 2010 and are predicted to reach nearly $58 billion in 2016 link. Successful antibody treatments, however, 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.

Protocols

Part 1: Prepare cell lysates

1. You will each have an ice bucket at your bench with the following pre-chilled items inside: two empty eppendorfs, RIPA buffer, protease inhibitors, and PBS. Begin by labeling the eppendorf tubes as K1 and xrs6 (plus your section).
2. Pick up your cell dish from the incubator in TC, and place it at a 30-45 degree angle tilted downward in your bucket.
3. Add 2.5 μL of protease inhibitors to your 250 μL of aliquotted RIPA buffer.
4. Aspirate the media from each well and add about 2 mL (very approximate!) of ice-cold PBS per well by pouring.
5. Obtain two pre-chilled scrapers from the fridge.
6. Aspirate the ice-cold PBS and repeat the wash once more – make sure to remove ALL of the PBS after this wash.
7. Add 100 μL of lysis buffer across the top of each well, allowing it to run down the well.
8. Collect the cells to the bottom of the well by scraping each well with a fresh cell scraper.
   • First rub back and forth with the scraper to coat cells with lysis buffer. Then go from top to bottom windshield wiper style to pool them down towards the bottom of the tilted dish.
9. Add the contents of each well to its respective eppendorf tube.
10. Incubate the eppendorf tubes on ice for 15 min.
    • Meanwhile, take two fresh eppendorf tubes and begin to chill them for a later step.
11. Spin the tubes at max speed in the cold room centrifuge for 15 min to pellet insoluble material. Bring your eppendorf tubes to the TA who will spin them for you.
12. Meanwhile, label a second set of eppendorf tubes as in Step 1 and chill.
13. Transfer the supernatant to the new set of eppendorf tubes and keep on ice – be careful not to disturb the pellet at the bottom!

Part 2: Measure protein content

You will now measure the total protein concentration in each cell lysate to determine the volume required to evaluate equal protein amounts by Western blot. We are using the Precision Red Advanced Protein Assay from Cytoskeleton.

1. Add 10 μL of cell lysate to 990 μL of Precision Red reagent in a cuvette: prepare K1, xrs6, and a blank "lysate" using your leftover RIPA buffer.
2. After 1 minute, measure each sample at 600 nm, using the RIPA sample as a blank.
3. Calculate the two stock protein concentrations using the following information
   • 1 absorbance unit = 100 ug protein/mL reagent / cm
   • the path length of the spec is precisely 1 cm
   • don't forget to account for the dilution factor
4. Next, calculate the volumes of lysate and water required to add 20 μg of total protein in each well of your SDS-PAGE gel in a total volume of 15 μL.
   • if your concentration is greater than 20 μg in 15 μL, use water to make up the remaining volume
   • if your concentration is less than 20 μg in 15 μL for at least one sample, scale both samples down to a lower amount, such as 10 μg

Part 3: Separate proteins by SDS-PAGE

Part 4: Transfer proteins to membrane

For next time

Reagent list

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