Monday, August 13, 2012

Keira- Final antibody tests (Week 7)

Last week I showed you photographs of larvae stained with the phosphohistone antibody. This week, I'll discuss my findings about two more fluorescent antibodies I tested. A few weeks ago, I discussed the process of fluorescent antibody staining; I'll give you a quick recap now. First, the (already fixed) larvae are soaked in a primary antibody. This antibody has been developed to bind specifically to certain proteins of interest. After rinsing the primary out, the larvae are soaked in a secondary antibody. This fluorescently labeled antibody binds specifically to the primary. When excited with laser light, the secondary antibody will fluoresce, thereby showing us the location of the primary antibody.

The first antibody that I will be showing in this week's post is a MAPK (mitogen-activated-protein-kinase) antibody. MAPK refers to a key signaling pathway through which the cell responds to external stimuli. The antibody I tested is called p44. This will tag a certain "flavor" of MAP's; this "flavor" is the "44" part of the name. The "p" denotes that this antibody will tag the phosphorylated proteins. That is, only the activated proteins that are actively involved in signaling will be shown.

The first picture shows a D.excentricus larvae that has been stained with the p44MAPK antibody. This gives us a baseline for what the antibody looks like and what kind of signaling it is involved with in a normal, untreated larva.

The next photograph shows us a larva that has been deciliated. This is achieved by soaking the larvae in seawater at two times its normal concentration, which sends the larvae into hypertonic shock and causes them to lose their cilia.

 The p44MAPK signaling pathway is clearly activated by the stress of deciliation. We see much brighter labeling along the ciliary band as well as some illuminated skeletogenic cells.

Another antibody I tested was a SAPK or stress-activated-protein-kinase. It is in the family of MAPK and is activated by a variety of environmental stress and growth factors, among other things.

This is a photograph of the untreated larvae labeled with the SAPK antibody. The edges of the epithelial cells are illuminated, giving the larva a lace- or spiderweb-like appearance.

This larva has been treated with the same deciliation process. The deciliation appears to activate the SAPK signaling pathway as well. The ciliated band is bright and well defined. As the majority of cilia-possessing cells are located in the ciliated band, it makes sense that labeling in the ciliated band would be brightest because the effects of deciliation would be more pronounced where most cilia are located.

1 comment:

  1. After reading this blog I came to know about Final antibody tests.
    Antibody Conjugate