Pkc Carraway Lab Report

Analysis In the experiment, the scientist observed a series of cells, the first one being cheek cells. After swabbing the inside of their cheeks and preparing a slide, the scientists were able to see the cytoplasm, nucleus and cell membrane of this undyed cell. While observing these cells under 400X, they noticed that the cheek cells varied in shape, some being almost perfectly spherical, while others resembled an oval figure. Additionally, these cells appeared to be grouped up and messily stacked on top of one another.

Based on the samples the expression of rGFP was the greatest in the E3 both quantitatively and qualitatively. The purity of rGFP was however low. The rGFP protein was purified using the Ni+2 agarose chromatography. This part of the experiment did not achieve a high purity. Causes of this may have incorrect pipetting mechanism or incorrect amount deposition. Improving pipetting skills might have lead to more accurate results. A better comprehension of the purpose of each step in the protocol may have resulted in improved results. Also personally having to use GCE of a partner’s could have altered the results. Being more strict with the lab procedures, and not messing up with the timings of certain steps by accident would have improved data. Although minor errors may have occurred the greatest fluorescence occurred in wash fraction W4 and elution fraction E3 quantitatively, and this data matched the qualitative observation of the fluorescence in both these samples. W4 had the greatest protein amount. W1 was the void volume and only contained breaking buffer without rGFP therefore there should have been no fluorescence, and this occurred correctly. Samples W2 and W3 had slight fluorescence which could be explained by the rGFP lacking the His6 tag required to bind to the Ni+2 agarose, or all the binding sites in the column could

To make the specimen compatible with both forms of advanced microscopy, they sufficiently prepared samples by coupling the specimen with a fluorescence that was also conductive. This technique was accomplished with the FlouroNanogold label, which contains gold nanoparticles covalently bonded to a fluorescence label. That way, the LM worked as well as the EM for the same set of kinetochores that were being studied. The Hec1 protein was stained in this case because this protein naturally delineates the structures to be studied.

One protein mentioned called RhoA is said to force cells into shape. Another protein called Rac1 can maintain myosin phosphorylation to put a controlling influence on entotic

The mechanism in which cells move either towards, or away from a stimulus is referred to as chemotaxis (1). Chemotaxis is initiated in eukaryotic cells when the cells first sense an external chemotactic gradient (2). The presence of this gradient is sensed by a class of receptors, called 7-transmembrane heterotrimeric G-protein coupled receptors (GPCRs) (2). As the name implies, these proteins are located in the plasma membrane of eukaryotic cells. This external gradient triggers an intracellular Phosphatidylinositol (3,4,5)-trisphosphate (PIP3) gradient to form (2). PIP3 is a phospholipid that is located in the plasma membrane of eukaryotic cells (3). This PIP3 gradient induces a signaling

The cells are placed into a flask and are forced through a nozzle so small that they must pass through one by one. In the nozzle, the cells are vibrated at different frequencies to produce drops (3). The drops of cells are then scanned by a laser that is used to count and measure each cell. Separating populations of cells involves attaching antibody linked fluorescent dye to certain cells of interest (3). The information that is gathered from the sorting and measuring of the cells is evaluated by a computer. The final steps for the FACS include applying an electrical charge to the drops of cells (3). Before the drop of cell forms at the end of the nozzle, a charge is applied to the stream that will determine where the drop will go (3). Based on the charge, the drip is either moved left or right with electrodes or placed in to designated final tubes. Quantifying the FACS information involves displaying the information so we know how many cells of each color and charge were

Proteins, as macromolecules, cannot directly diffuse through the pathway of NPCs due to the presence of the disordered region of channel nucleoporins; the bundles of the channel nucleoporins are compactly aligned in disarray in the central pore, and certain phenylalanine-glycine (FG) repeats, which are present on the bundles, are believed to associate via low-affinity, cohesive interactions to form a permeability barrier of the pore (Xu & Powers, 2013) and stop macromolecules to pass through freely, thus it requires energy input and aids from other molecules to traffic proteins through NPCs.

6- Outer Plexiform Layer :where photoreceptor cells form connections with the bipolar and horizontal cells of the inner nuclear layers.

This indicates that there is either some other pathway functioning redundantly to restore P granule segregation or that NPP-19 is restoring proper P granule segregation, but does not control the distribution of other cytoplasmic determinants like PIE-1.

It is action is mediated by Protein Kinase G (PKG) , a cGMP-dependent protein kinase, that phosphorylates target proteins in the cell.

attracting the lipid kinases to IRS proteins, the reaction moves the kinases to PIP 2 located on the

The movie is combined for 2 different events shown in Figure 3 – Figure Supplement 1 and 2. The movie shows increased flexibility of the microtubule growing beyond the point where KIF21B was stalled (top panel, upper MT) and repeated short excursions from the point of KIF21B stalling (bottom panel). Experiment was performed in the presence of mCherry-EB3 (20 nM) and Rh-tubulin (0.5 µM) with KIF21B-GFP (0.5 nM). The movie consists of 128 frames acquired in a time-lapse mode. Scale bar, 2

Cameleons, the first generation of GECIs [161], are chimeric proteins that, in their original iteration, were composed of a blue or cyan FP as a FRET donor and a green or yellow FP as a FRET acceptor. The two FPs are connected by CaM and its binding partner M13 (the CaM-binding domain of myosin light chain kinase) (Figure 6A). A similar design with only the CaM-binding domain was also reported in 1997 [162]. When the CaM domain of a cameleon binds to Ca2+, it changes its conformation and sequentially binds to M13, which results in a more compact conformation that decreases the distance between the FRET donor and acceptor. This decreased distance causes an increase in FRET efficiency that manifests itself as

BiFC is a technique to test for interactions between two proteins by fusing different fluorescent fragments to the two putative partners. If the proteins in fact bind, fluorescence will be observed as the two fragments are brought into close proximity. Yolk platelets make Xenopus cells highly autofluorescent, so we will use a variant of enhanced yellow fluorescent protein (EYFP) called VENUS that matures more rapidly than EYFP and is more resistant to pH. We will cleave VENUS at position N144 as used in previous Xenopus BiFC experiments29,30. We will amplify each fragment by PCR and clone into the expression vector pCS2+. We will then fuse one of these fragments to the N-terminus of FMRP and the other fragment to the 5’ end of Cyclin E mRNA, amplify our constructs by PCR, and check the constructs by sequencing. We will

Based on the supramolecular architecture of rhodopsin captured by the AFM image and available crystallographic data, in 2003 the first model of higher order organization of a GPCR in its native membrane was derived implicating involvement of transmembrane domains in contact formation between neighboring rhodopsin molecules within the dimer as well as between distinct rows of dimers (Liang et al., 2003). This model suggested that the intradimeric contacts defining primary dimer interface involve transmembrane helices 4 (TM4) and 5 (TM5), whereas the formation of rhodopsin dimer rows is facilitated by the interaction between helices 1 (TM1) and 2 (TM2). Interestingly, a structure of squid rhodopsin, solved soon after, agreed with TM4/TM5 dimer very well (Murakami and Kouyama, 2008). Different experimental strategies have been further applied to identify rhodopsin dimer interface to validate this semi-empirical rhodopsin-packing model. Firstly, cysteine mutagenesis of specific amino acids predicted by molecular modeling to be involved in formation of the dimer-contacting surface followed by intermolecular crosslinking in the presence of cupric orthophenanthroline (CuP) let to the identification of two amino acids Trp175 and Tyr 206 at the dimer interface of opsin heterologously expressed in membranes of COS1 cells (Kota et al., 2006b). Trp175 is present in the extracellular loop connecting TM4 and TM5 and Tyr206 is located on the extracellular side of TM5 therefore