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Homework answers / question archive / Antibody probing Specific antibodies (called primary antibodies) can detect antigens (proteins attached at the surface of the membrane)

Antibody probing Specific antibodies (called primary antibodies) can detect antigens (proteins attached at the surface of the membrane)

Chemistry

Antibody probing Specific antibodies (called primary antibodies) can detect antigens (proteins attached at the surface of the membrane). These antibodies are raised in a variety of species, including mouse and rabbits. Antibodies contain two identical variable domains that bind to the antigen and are responsible for the antibody-speci?city. Antibodies also contain a constant domain that determines the class of antibody (IgG, A or M for example). Within a class of antibodies, the constant domain will vary from species to species. Therefore, secondary antibodies directed against the constant domain of rabbit IgG or mouse IgG can be raised by injecting these domains into different species (goat or chicken for example). Purified secondary (and primary) antibodies can be conjugated to a reporter molecule allowing for the visualization of antigen-antibody complexes. The reporter molecule can be an enzyme that catalyzes the conversion of a chromogenic substrate into a precipitating product or a luminescent substrate. In the ?rst case, the colored product precipitates on top of the antigen- primary &secondary antibody complex. In the second case, light is emitted from the region of the membrane containing a high concentration of the antigen-primary &secondary antibody complex, and can expose an X-ray film placed into contact with the membrane. The position of the antigen is detected after development of the ?lm. In this laboratory exercise we use a secondary antibody that is conjugated with a type of fluorophores characterized by an emission spectra in the infrared range. It is possible with this system to detect two different antigen-primary &secondary antibody complexes on the same membrane because you can select between two distinct Infrared ?uorophores (See Fig. A2). The antigen-primary &secondary antibody complex is detected by scanning the membrane with two diode lasers. The absorption maxima of the two ?uorophores: [RDye ?uor 700 (or Alexa Fluor 680 nm) and IRDye-SOO closely match the 700 and 780 nm excitation wavelength of the two lasers. Mil-an one lmmm mum Ina. I ?u. mu no out Uni-m Il? um Inga I: mm. mm mm- meow In as i GBSESaSS Hm E Millikan-nu aaaaasezzis

Figure A2: Absorption and emission profiles of the two infrared fluorophores conjugated to the secondary antibodies. In this lab exercise, we use only one primary antibody and thus we only need a single secondary antibody. The primary antibodies that detect the TAP tag (anti-TAP) are purified rabbit polyclonal antibodies. The secondary antibody used is an anti-rabbit IgG conjugated to IRdye-700 nm. The principle of the antibody detection with two primary and two secondary antibodies is illustrated in Fig. A3. anti-P1 anti-P2 (Rabbit) (mouse) 10 Ab/Ag complex Membrane Ag P1 P2 P3 Incubation with P1 P2 P3 primary antibodies Incubation with anti-rabbit IgG secondary antibodies anti-mouse IgG 2"Ab/1"Ab /Ag complex P1 Dual laser scanning P2 (Infrared) 700 nm P1 P2 P3 800 nm pa Figure A3: Co-detection of proteins transferred onto a nitrocellulose membrane. Antibodies directed against PI and P3 (anti-P1 and anti-P3) bind to Pl and P3 to form antigen- primary antibody complexes (1Ab/Ag). Secondary antibodies, that recognized specifically the anti-mouse IgG or anti-rabbit IgG, are then used and form antigen-primary & secondary antibodies complexes (2"Ab/1* Ab/Ag). The secondary antibodies are conjugated to two distinct fluorophore molecules allowing the visualization of the 2"Ab/1 Ab/Ag complexes and the amplification of the signal since more there are several molecules of 2Ab bound to each molecule of 10Ab. Note there is another level of signal amplification since an antigen binds more than 1 molecule of 10 antibody.

APPENDIX B: Prestained protein ladder 4-20% Tris-Glycine Gel 5 pl/lane k Da* ~ 190 - ~ 120 - ~ 85 - ~ 60 - ~ 50 - ~ 40 - ~ 25 - ~ 20 - ~ 15 - ~ 10 - * Coupling of the chromophores to the proteins affects their apparent molecular weight in SDS-PACE relative to unstained standards. Each band in the pre-stained ladder is calibrated against the unstained BenchMark" Protein Ladder on a 4-20% Tris-Glycine gel and the apparent molecular weight is reported on the product profile. The pre-stained protein ladder should only be used to determine an approximate size molecular weight. Note: The Membrane scanner is very sensitive to blue and the ladder (except the pink band) appears on the membrane scan.

Additional information 1) You were given two clones where the sequences for components of the PDH complex are TAP tagged: - Clone YER178W (PDA1): the sequence coding for the a-subunit of El is tagged. - Clone YFLO18C: (LPD1): the sequence coding for the E3 polypeptide chain is tagged. 2) The primary antibody used is a rabbit antibody that specifically recognized the calmodulin-binding peptide (part of the double tag) 3) The secondary antibody was a anti-rabbit IgG made in goat and labeled with a fluorescent molecule 4) The full tag adds 20 kDa to the tagged protein The molecular weights of the untagged proteins are: a-subunit of E1: 42.7 kDa E3: 51.5 kDa Lane 1: Clone A, IgG beads Lane 2: Clone A, CAM beads Lane 3: Clone B, IgG beads Lane 4: Clone B, CAM beads 2 3 Figure 1: Immunoblot The stars (*) represent the bands of interest, and the question mark (?) represents a reactive band that does not correspond to any protein from the affinity purified complex.

Questions 1) Based on the data from the immunoblot (figure 1), assign the name of a clone (YER 178W or YFLO18C) to each unknown clone (A and B). Provide a detailed explanation based on the identification of the proteins labeled by stars. (10 pts) Important: the proteins labeled with a star are different or represent different forms of the same protein. In your explanation make sure the grader knows which starred protein you are referring to (indicate the corresponding lane to avoid any ambiguity). 2) The antibody selected was an antibody that recognized the calmodulin binding peptide. Explain why an antibody recognizing the other affinity tag (protein A) was not selected? (4 pts)

3) In any study controls should be implemented to exclude false positive and false negative. a) What would be the interest of repeating the tandem purification on a wild-type yeast strain expressing no tag protein? (4 pts) b) The results in Figure 1 shows that the tagged proteins were affinity puri?ed. What type of antibody detection would you propose to demonstrate that the affinity purified protein was part of the PDH complex and not free in the cytosol? (4 pts) 4) Suggest a possible identity for the protein labeled with a question mark (?) on figure 1. Brie?y explain your reasoning. (3 pts)

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