Antibody staining - lab report PDF

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Introduction: Antibody staining is procedure that is used in laboratory to stain a sample of antibody .the goal of this procedure is to detect the presence of certain antigens that is founded in a certain protein. Also, it is used to follow the pathway of certain antibody in vivo, so that they can be clearly viewed. In many techniques, people use a secondary antibody that has a fluorescent tag. Attached, in this a secondary antibody will attaches. Itself to a primary antibodies in the sample, and fluoresce under light to view the complex. There are many techniques for anybody staining as a general review; immunohistochemistry technology is used to stain antibodies and other types of proteins, nucleic acids signaling molecules. Immunohistochemistry staining methods can be classified as immunofluorescence, immunoenzymological staining and affinity histo-chemistry.

According to different types of staining procedure, IHC can be divided into subtype depending on how many steps are used. In our lab experiment, we used several types of staining. These staining are as following: 1. Anti-histone H3 (mono methyl K4) antibody-ChIP great (ab8895). 2. Chicken anti-rabbit IgG (H+L) secondary antibody alexa flouro 488 conjugate. 3. Goat anti-mouse IgG2a secondary antibody, alexa flouro 555 conjugate. 4. Anti-acetylated alpha tubulin antibody.

Procedure: Each experiment has different procedure, but they are quite similar from the way to staining for anti-histone H3, the important is to have a full fixation of the antibody followed by staining to view it. The procedure as following: 1. A sample of cells is plated onto glass coverslips and fixed fo 5070% confluence with 4% with paraforma-1dehyde in PBS minutes. 2. Antibodies are diluted in PBS and then applied 20ul drops on Para film. 3. Incubate for 60 minute at room temperature. 4. Cell is washed 3x with PBS during incubation followed by staining with secondary antibodies.

5. The solution of staining is 1x PBS/90% glycerol containing 1 mg/ml paraphenylendiammine and 1 ug /ml DAPI. The procedure of goat-anti mouse IgG2a secondary antibody is quite different. Since we use secondary antibodies. The procedure as following: 1. Centrifuge the protein solution for 5 minute on high speed. 2. Add only the supernatant to the experiment. 3. Dilute the antibody for a final concentration 1_10ug/ml.

Result of the experiment and discussion: For H-3 staining it indicates the function of histone_3. It shows that during DNT replication, the DNA accessibility is regulated via a complex – set of post –translational modifications of histones, also called histone code, and nucleosome remodeling. For anti-acetylated alpha tubulin antibody, the result of staining shows microtubules are required in function of cell such as movement, of chromosome during mitosis and meiosis, intracellular transport, establishing the morphology of cell.

Larva stained with Dapi

larva 1 _h3k4 staining

larva 1 a-tubulin staining

Reference: 1. http://www.immunohistochemistry.us/immunohistochemistrystaining.html

2. http://www.abcam.com/acetylated-alpha-tubulin-antibody-6-11b-1ab24610.html 3. https://www.lifetechnologies.com/order/genomedatabase/antibody/Mouse-IgG2a-Secondary-AntibodyPolyclonal/A-21137...