20.109(S14):Initiating transcript and protein assays (Day5)

Introduction

There are several ways to assess protein concentration, either quantitatively or as present above some threshold value. In the second module, you used one approach to identifying a specific protein that is only one among many in a complex mixture: Western blot. In this third module, you will use an approach that is somewhat easier to make quantitative: ELISA.

As you may recall, 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. For example, conformation-dependent antibodies are useful for distinguishing different collagen types.

As you learned about during Module 2, antibodies can be raised in animals (polyclonal) or special cell lines (monoclonal); they can also be genetically engineered. For example, 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 such as a mouse. 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 saw during Module 2 and will see again below.

Schematics of indirect and sandwich ELISA. Triangles indicate the protein of interest, and * indicates a conjugated enzyme for later detection. (Blocking step not shown.)

Today you will use antibodies against collagen in an indirect ELISA assay. Both indirect and sandwich ELISA are shown in the figure at right. Can you see why sandwich ELISA might be the superior assay with respect to sensitivity and specificity? In indirect ELISA, the first step is to bind protein extracts, in this case obtained from your two different culture conditions, to well plates. Next you will add a primary antibody that recognizes a particular antigen – namely, epitopes on collagen I or collagen II – to the sample wells. (Actually, before adding the antibody you will "block" the plate with milk protein to prevent non-specific binding of the antibody.) Next, any excess antibody must be washed away with a mild detergent. Finally, a secondary antibody – one that recognizes the primary antibody – must be added. The secondary antibody is conjugated to alkaline phosphatase, which will undergo a colorimetric reaction in the presence of its substrate. Thus, the relative quantity of protein can be assessed by absorbance spectroscopy following substrate addition. To quantify the absolute amount of protein, you will run dilutions of a collagen standard in parallel with your culture samples.

During your ELISA incubation steps, you will prepare three qPCR reactions for each cDNA mixture that you prepared last time. The first two reactions will use primers for our genes of interest: collagen II and collagen I. The final reaction will be a control for cDNA amount that contains primers for the 18S ribosomal RNA gene. The culture condition that has relatively higher collagen II and relatively lower collagen I, taking into account amount loaded based on 18S results, should be the culture with more chondrocyte-like cells.

Reference: Abbas, A.K. & Lichtman, A.H. (2005). Cellular and Molecular Immunology (5th ed.). Philedelphia: Elsevier Saunders.

Protocols

Part 1: Day 1 of ELISA

ELISA protocol

We will run this assay in a 96-well microtiter plate and make measurements using an absorbance plate reader.

1. Spin your pepsin-digested samples from last time in the microfuge for 5 minutes at maximum speed (16,000 rcf).
2. Label one 96-well plate for your collagen I assay, and one for your collagen II assay.
   • Separate plates will make it convenient to keep track of antibody addition and to later have different reaction stop times if necessary.
   • You may want to label the lids in full, and the bottoms of the plates in shorthand. Or just be careful not to mix up the lids later!
3. The first step in indirect ELISA is to adsorb all your samples to the wells. You will also need to prepare standard samples in the same plate, which get treated just the same as your test samples. These standards will be used as a reference for protein concentration. Both standards and unknown samples will be run in duplicate (see figure at right).

   

   Suggested ELISA plan. This plan can be used for both your collagen I and your collagen II plate. In each case, columns 1 and 2 are duplicates of the collagen standards, and column 3 contains your experimental samples and a few wells (labeled BLANK) to measure background.

   • Super-groups may want to join together on one plate per collagen, to reduce experimental error/variation from using two different standard curves. However, be sure to keep some BLANK wells. For example, the second group could use column 4 wells A-D.
4. You will be given 250 μL aliquots of collagens I and II at 10 μg/mL each. Prepare your standards as follows:
   • Ultimately, you want to pipet 50 μL per well of each standard concentration, two wells per standard.
   • Option 1:
     • Per collagen, prepare 7 eppendorf tubes with 120 μL of PBS each.
     • Add 120 μL of the 10 μg/mL collagen to the first eppendorf tube, and vortex.
     • Now take 120 μL of that standard (now 5 μg/mL) and add it to the next eppendorf tube.
     • When you are all done or as you go, pipet the standards into the appropriate wells.
   • Option 2:
     • Pipet 50 μL of PBS into wells 1 and 2 of rows B-H (skip A!).
     • Pipet 100 μL of the 10 μg/mL collagen into the appropriate wells (A1 and A2).
     • Using a regular or multichannel pipet, transfer 50 μL of these solutions to the next wells down (B1 and B2), and mix with the PBS.
     • Repeat, now moving 50 μL of the 5 μg/mL solution in the B wells down to the C wells.
   • Either of these methods is called making doubling dilutions. Which way do you think introduces less error? Which way do you think is faster?
5. Now add 50 μL of your samples to the appropriate wells. For the blank wells you should add PBS.
6. Cover each plate (CN I and CNII) when you are done, wrap around it with parafilm to better prevent evaporation, and allow the samples to sit for 80 minutes. In the meantime, set up your qPCR reactions (Part 2).
   • Ask the teaching faculty for assistance if you have not used parafilm before. Note that it is stretchy.
7. After the incubation time has passed, you will wash and then block your plate.
   • First, flick the solutions in the plate into the sink.
   • Using the multichannel pipet and a reservoir, add 200 μL of Wash Buffer to each well, then gently swirl the plate (by hand) for a few seconds.
   • Flick the solutions out again, and then blot the plate against paper towels. You can smack the plates pretty hard, but it is possible to break them!
   • Repeat the wash one more time.
   • Finally, add 200 μL of Block Buffer to the plate. Wait another 60-90 minutes. In the meantime, you can work on cell viability analysis (first listed on Day 4 Part 4).
8. Repeat the wash step that you performed above, again with two rinses.
9. When you are ready, ask the teaching faculty for some primary anti-collagen antibodies (these should be diluted at the last minute). Add 100 μL of diluted antibody per well.
10. Your samples will be left overnight in antibody solution, then moved back to block buffer by the teaching faculty.

Part 2: Set up qPCR reaction

Important background, do not skip

Each group will have 6 samples in duplicate, or 12 reactions in total. In the schematic below, the top row indicates the group colour (obviously!). The next row indicates the sample identity as X-Y, where X is the culture condition (keep track of what is 1 and 2) and Y is the replicate number. The final row indicates the gene of interest and thus the primer set that will be used.

Ultimately, you will add your cDNAs to the appropriate wells in your row. Then, the teaching faculty will add the appropriate master mix (containing water, primers, SYBR green dye, buffer, etc.) to all of the wells with a multi-channel pipet. We are doing most of the preparations for you today for a few reasons. Regarding the master mixes: qPCR is very sensitive, so we want to reduce error and any differences between samples as much as possible. Regarding the plating: note that the DNA-binding dye is very light-sensitive, so we want to do this step quickly rather than one group at a time. (Similar reasoning about reducing pipetting variability in the plating step also applies.)

Plating plan for qPCR assay, T/R.

Plating plan for qPCR assay, W/F.

Preparation

1. For each culture condition, you need to prepare enough diluted cDNA for 6 reactions, plus some extra. Unless you had really low RNA yield (talk to the teaching faculty if so), you should prepare the following as your "1x" stock of cDNA, times 7 reactions: 1.9 μL of cDNA plus 3.1 μL of water (so 13.3 plus 21.7 μL, respectively).
   • We normally prepare qPCR reactions with slightly different volumes than we'll ask you to use today (long story...), so for my ease of comparison later as I revise the module and see what works well, you will consider the above cDNA 1x or undiluted stock.
2. The primer sets and cycling conditions have been validated for a wide range of starting cDNA concentrations. Decide what you want to use according to the criteria below, and for each condition prepare 30 μL plus at least 10-20% excess of your dilution (in nuclease free water).
   • If you had 500 ng (or close to it) of RNA in your cDNA reaction, a 5-10x further dilution of your cDNA should work well.
   • If you had a bit less, using the stock directly or a 2x dilution may be more appropriate.
3. Add 5 μL of your cDNAs to your 12 wells according to the schematic above and check your group colour off the list.
4. When everyone is done, the teaching faculty will add the three different master mixes, and run the plate over to the BioMicro center shared equipment facility in building 68.
   • Groups that happen to have down-time when the plate is ready may come and observe the plate set-up.

Part 3: Continue viability analysis (optional)

Assuming you didn't have a chance to finish (or even start!) the viability analysis last time, see Day 4 Part 4 and start today. You should have downtime next time as well to finish.

Part 4: Research idea discussion

During an incubation step today, take some time to discuss the five research results you wrote up for homework with your lab partner, guided by the instructions below.

Writing a research proposal requires that you identify an interesting topic, spend lots of time learning about it, and then design some clever experiments to advance the field. It also requires that you articulate your ideas so any reader is convinced of your expertise, your creativity and the significance of your findings, should you have the opportunity to carry out the experiments you’ve proposed. To begin you must identify your research question. This may be the hardest part and the most fun. Fortunately you started by finding a handful of topics to share with your lab partner. Today you should discuss and evaluate the topics you’ve gathered. Consider them based on:

• your interest in the topic
• the availability of good background information
• your likelihood of successfully advancing current understanding
• the possibility of advancing foundational technologies or finding practical applications
• if your proposal could be carried out in a reasonable amount of time and with non-infinite resources

It might be that not one of the topics you’ve identified is really suitable, in which case you should find some new ideas. It’s also possible that through discussion with your lab partner, you’ve found something new to consider. Both of these outcomes are fine but by the end of today’s lab you should have settled on a general topic or two so you can begin the next step in your proposal writing, namely background reading and critical thinking about the topic. Check in with Reggie and get his feedback about your ideas for a few minutes before leaving today.

A few ground rules that are 20.109 specific:

• You should not propose any research question that has been the subject of your UROP or research experience outside of 20.109. This proposal must be original.
• You should keep in mind that this proposal will be presented to the class, so try to limit your scope to an idea that can be convincingly presented in a twelve minute oral presentation.

Once you and your partner have decided on a suitable research problem, it’s time to become an expert on the topic. This will mean searching the literature, talking with people, generating some ideas and critically evaluating them. To keep track of your efforts, you should start a wiki catalog on your OpenWetWare user page. How you format the page is up to you but check out the “yeast rebuild” or the “T7.2” wiki pages on OpenWetWare for examples of research ideas in process. As part of a later FNT assignment, you will have to print out your wiki page specifying your topic, your research goal and at least two helpful references that you’ve read and summarized.

For next time

1. Begin to define your research proposal by making a wiki page to collect your ideas and resources (you can do this on one page with your partner or split the effort and each turn in an individual page). Keep in mind that your presentation to the class will ultimately need:

• a brief project overview
• sufficient background information for everyone to understand your proposal
• a statement of the research problem and goals
• project details and methods
• predicted outcomes if everything goes according to plan and if nothing does
• needed resources to complete the work

You can organize your wiki page along these lines or however you feel is most helpful. For now, focus on coming up with a research problem and giving a little background about it. Print your user page(s) for next time, making sure it defines your general topic, your specific idea, and two or more references you've collected and summarized. Keep in mind that you're not committed to this idea - if you come up with something that you like better later on, that's fine.

After this one, there will be no more assignments to hand in besides the two remaining major assessments. However, by the end of Day 6 or so (whether in or outside class), you should meet with someone from another group to discuss your research proposal. See Part 4 of Day 6 for a description of how to proceed and the Day 6 Talk page for the randomized list of pairings. (You and your partner should meet separately with people who are not themselves in the same group, so that you each get a chance to give your take on the project.)

Reagent list

• qPCR reagents
  • 2X Power SYBR Green Master Mix from Applied Biosystems
  • Collagen II primers, 100 nM
  • Collagen I primers, 100 nM
  • 18S primers, 25 nM

• ELISA solutions
  • PBS reconstituted from EMD tablets
    • 140 mM NaCl
    • 10 mM phosphate buffer
    • 3 mM KCl
    • pH 7.4
  • Wash buffer
    • PBS
    • 0.05 % Tween 20
  • Block buffer
    • PBS
    • 5 % powdered milk

• ELISA collagen-specific reagents
  • All from GeneTex
  • Collagen I standard, diluted from 1 mg/mL in PBS
  • Collagen II standard, diluted from 1 mg/mL in PBS
  • Collagen I antibody, diluted 1:4000 from 1 mg/mL in PBS
  • Collagen II antibody, diluted 1:4000 from 1 mg/mL in PBS

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