20.109(S23):M1D5

Introduction

You will use commercially available antibodies to identify the 6xHis tag on the MAX protein in your experiment. The ability to bind specific proteins using antibodies, or immunoglobulins, is critical in immuno-fluorescence labeling 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 6xHis tag on the MAX protein. 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 fluorescent molecule emits at 800 nm that will enable us to visualize our specific protein from the non-specific proteins that are in the purified MAX-6xHis solution.

Protocols

Part 1: Probe blot for Western blot experiment

To ensure the steps included below are clear, please watch the video tutorial linked here: [Western Blot]. The steps are detailed below so you can follow along!

1. The blot that you prepared in the previous laboratory session was stored in the 4 °C cooler in blocking buffer.
2. Retrieve your blot from the front laboratory bench.
3. Prepare the primary antibody solution.
   • Dilute the primary antibodies in 10 mL of blocking buffer.
   • To probe for the MAX protein you will use a rabbit anti-α-MAX primary antibody at a 1:1500 dilution.
   • To probe for the 6xHis tag you will use mouse anti-α-His antibody at a 1:1000 dilution.
4. Pour off the blocking solution in the sink.
5. Add the primary antibody solution to your blot.
6. Carefully place the blot on the rotating table and cover with aluminum foil.
7. Shake at 65 rpm for 60 min.
8. Obtain your blot from the rotatating table and pour the primary antibody solution into a conical tube.
   • Label with team and section information.
   • Because the antibody is in excess, the diluted primary solution can be re-used and is worth saving until you see the results of your Western blot.
9. Add enough TBS-T to cover the membrane.
10. Return your blot to the rotating table for 5 min at 80 rpm.
11. Repeat for a total of 3 washes.
12. Immediately before pouring off the last wash, prepare the secondary antibody solution.
    • Dilute the secondary antibodies in 10 mL of blocking buffer.
    • To visualize the α-MAX (raised in a rabbit) you will use the donkey anti-rabbit IR680 (GREEN) antibody at a 1:10,000 dilution.
    • To visualize the α-His (raised in a mouse) you will use the goat anti-mouse IR800 (RED) antibody at a 1:10,000 dilution.
    • The secondary antibodies are light sensitive and should be kept in the dark! Use aluminum foil to wrap your tube.
13. Add the secondary antibody solution to your blot.
14. Carefully place the blot on the rotating table and cover with aluminum foil.
15. Shake at 65 rpm for 60 min.
16. Pour off the secondary antibody in the sink.
17. Wash the membrane by adding TBS-T and shake for 5 min at 80 rpm, using the rotating table.
18. Repeat for a total of 3 washes.
19. The ChemiDoc Imaging system is located in the Niles laboratory, one of the teaching faculty will accompany you there to image your blot.

Part 2: Draft Data summary slide for MAX-6xHis purity and concentration results

To get a headstart and further feedback from the Instructors, you will draft the slide that will present the MAX-6xHis purity and concentration data for your Data summary today in class. With your partner, use the template below, the Instructor comments from your previous homework assignments, and the helpful hints from the Comm Lab workshop to craft a data slide with the required elements.

Individually you and your laboratory partner each crafted figures (with a title and caption) using the MAX-6xHis purity and concentration data. In this exercise you will come together to decide how to best present these data for the Data summary!

Reagents list

• Odyssey blocking buffer (from Licor)
• rabbit α-MAX primary antibody (from ThermoFisher)
• mouse α-His primary antibody (from Sigma)
• donkey α-rabbit IR680 secondary antibody (from Licor)
• goat α-mouse IR800 secondary antibody (from Licor)
• ChemiDoc Imaging system (from BioRad)

Navigation links

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