20.109(F19):Complete gamma-H2AX assay and prepare CometChip (Day3)

Introduction

The ability to bind specific proteins using antibodies, or immunoglobulins, is critical in immuno-fluorescence labeling and Western blot analysis. Antibodies are typically 'raised' in mammalian hosts. Most commonly mice, rabbits, and goats are used, but antibodies can also be raised in sheep, chickens, rats, and even humans. 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. 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. Antibodies can be raised not only to detect specific amino acid sequences, but also post-translational modifications and/or secondary structure. Therefore, antibodies can be used to distinguish between modified (for example, phosphorylated or glycoslyated proteins) and unmodified protein.

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. In this process, normal antibody-producing B cells are fused with immortalized B cells, derived from myelomas, 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 over time with no intervention, so ultimately only the fused cells, called hybridomas, remain. A fused cell with two nuclei can be resolved into a stable cell line after mitosis.

Generating monoclonal 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 the purity (that it has few other antibodies), the specificity (that it recognizes the antigen and not other spurious proteins) and the concentration (sometimes called 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. The purity, specificity and titer of the new antiserum will likely differ from those 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 polyclonal antibodies.

In your experiment, you will use a primary antibody to bind the γH2AX foci. Then a secondary antibody will be used that is specific to the conserved region of the primary antibody. The use of secondary antibodies allows researchers to tag the primary antibody. In our assay, the tag is a 488 nm fluorescent dye that will enable us to visualize double-strand breaks via microscopy. As a reminder, during the last laboratory session you treated cells with two concentrations of either H₂O₂ or MMS for the H2AX assay. Since then, the teaching faculty fixed all the wells with paraformaldehyde. Your task for today is to permeabilize the cells, which will enable the antibodies to enter the cells and bind γH2AX.

Protocols

Part 1: Complete antibody staining for γH2AX assay

Immunofluorescence staining chamber

At this point in the assay sterility is no longer a concern and you will complete the following steps in the main laboratory at your bench.

1. Obtain your 12-well plates from the front laboratory bench.
2. Gather an aliquot of 1 X TBS from the front laboratory bench.
   • Prepare 1.2 mL solution of 0.2% Triton X-100 (v/v) in 1X TBS in a micro centrifuge tube. Triton stock (10%) is at the front laboratory bench.
   • Prepare 2.5 mL solution of 1% BSA (v/v) in 1X TBS in 15ml conical tube. 10% BSA stock is at the front bench.
     • One of the preparations will be the blocking solution used in Step #8 and the other preparation will be used in Step #9 for the primary antibody solution.
3. Obtain a staining chamber from the front bench and add a damp paper towel to each side of the parafilm. Label parafilm with experimental details.
4. Obtain a fine gauge (26 3/8) needle and a pair of tweezers from the front laboratory bench.
   • Carefully press the tip of the needle against the benchtop to bend it into a right angle such that the beveled side of the needle is the interior angle.
5. Use the 'hook' created with the needle to lift the coverslip from the bottom of the well, then use the tweezers to 'catch' the coverslip.
   • Practice plates with coverslips will be available at the front laboratory bench.
6. When you are confident with your ability to retrieve the coverslips from the wells, move one coverslip from each condition from your 12-well plates to the staining chamber. Cell-side UP!
   • The cell-side of the coverslip is the side that was facing up in the well of the 12-well plate.
7. Quickly permeabilize the cells by adding 150 μL of the 0.2% Triton X-100/TBS solution to each coverslip and incubate for 10 min at room temperature.
8. Aspirate the 0.2% Triton X-100/TBS solution and add 150 μL of BSA blocking solution to each coverslip, then incubate for 60 min at room temperature.
9. With 15 min remaining of the blocking solution incubation, prepare the primary antibody.
   • Dilute the mouse anti-γH2AX antibody 1:1000 in the 1.2 mL aliquot of BSA blocking solution.
10. Aspirate the block solution and add 150 μL of the diluted primary antibody solution to each coverslip before moving the next. Do not let the coverslips dry!
11. Carefully move your staining chambers to the 4 °C cooler.

Your samples will incubate at 4 °C in the primary antibody solution for ~48 h. The teaching faculty will replace the primary antibody solution with the secondary antibody solution, Alexa Fluor 488 goat anti-mouse diluted 1:200 in blocking solution along with Alexa Fluor 594 Phalloidin diluted 1:500, 1 h prior to the next laboratory session.

Part 2: Prepare CometChip

1. Obtain a sheet of gelbond film from the laboratory bench at the front of the room. The paper is protecting the hydrophilic side of the gelbond film.
   • Be sure to keep the paper associated with the gelbond film so you know which side is which.
2. Also obtain a special permanent marker from the front bench (Secureline Marker II).
   • If you use a marker from your drawer the ink will wash off during a later step in the CometChip assay protocol.
3. Use the ruler in your team drawer and the Secureline permanent marker to draw a 6.5 x 4.5 cm rectangle near the center on the hydrophobic side of the gelbond film. Label three rows--'A', 'B', and 'C'--along the outside of the short side of the rectangle.
   

   

   GelBond marking for CometChip
   • Note: you are writing on what will be the bottom of the CometChip and may want to write backwards so the labels are clear when you look at the top of your CometChip.
4. Prepare 20 mL of 1% normal melting point (NMP) agarose. Be careful as the agarose solution will be very hot!
   • Calculate the amount of NMP agarose powder needed for a 1% w/v solution. Check your math with the teaching faculty before you continue.
   • Obtain a small milk bottle from the front bench.
   • Weigh out the appropriate amount of NMP agarose and add it to the milk bottle.
   • Use a cylinder to measure 20 mL of 1x PBS and add it to the milk bottle with the NMP agarose powder.
   • Swirl to mix.
   • To melt the NMP agarose, microwave the solution for 20 seconds, swirl, then microwave for 3-second intervals until all crystals are in solutions. After each interval, remove the milk bottle and gently swirl while checking for unmelted agarose crystals. It is important that the solution does NOT boil as you will lose water to evaporation and the density of the agarose will be altered. If your solution starts to boil, immediately remove it from the microwave and gently swirl.
   • When no more crystals are visible in the solution take the milk bottle to your bench.
5. Obtain a small rectangle dish (labeled "scraped lid") and the CometChip 'stamp' from the front bench.
6. Add 2.5 mL of the agarose solution to the small dish, then quickly place the gelbond film in the dish with the marked hydrophobic side down. Remove the paper from the film.
7. Add 13 mL of the agarose solution on top of the gelbond film.
8. Slowly place the CometChip stamp on top of the agarose.
   • Lower the bottom left of the stamp first, then slowly allow the stamp to 'roll' into the agarose. Be sure to leave the top right corner of the small dish accessible.
   • Be careful not to introduce bubbles into the agarose and work quickly as the agarose will solidify as it cools.
9. Allow the agarose to solidify, undisturbed, on your bench for 30 min.
10. Add ~5 mL of 1x PBS to the small dish that contains your agarose CometChip.
    • Pipet in the 1x PBS using the accessible corner.
11. Slowly pull from one corner of the stamp to lift it away from your CometChip in the dish.
    • If the CometChip sticks to the stamp, carefully peel it off using tweezers.
    • Discard the PBS in the sink.
12. Remove excess agarose from the perimeter of your CometChip using a razor blade (obtain and return razor blade to front bench).
13. Clean the agarose from the bottom of your CometChip (gelbond side) using a Kimwipe.
14. Place your CometChip in the small dish containing 1x PBS for storage at 4 °C until next time.
    • Be sure the chip is completely submerged.
15. Please return the stamp to the front bench. Be sure never to wipe the stamp, as that will ruin the microposts.

Reagents list

γH2AX:

• permeabilization buffer: 0.2% Triton in Tris buffer saline (TBS, Invitrogen)
• blocking buffer: 1% bovine serum albumin (BSA) in TBS (BSA, sigma)
• 1:1000 primary antibody to γH2AX, mouse (Millipore)
• 1:200 Alexa Fluor 488 goat anti-mouse IgG (ThermoFisher)
• 1:500 Alexa Fluor 594 Phalloidin (ThermoFisher)
• Mounting media ProLong gold with DAPI (ThermoFisher)

CometChip:

• agar, normal melting point (Invitrogen)
• phosphate buffered saline (VWR)
• GelBond film (Lonza)
• 1 well dish (VWR)

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