Characterisation Of Gst Grp1 Ph Essay

1231 words - 5 pages

Characterisation of GST-GRP1PH
GST-GRP1PH was characterized using SDS–PAGE with sensitive Coomassie stain followed by automated in-gel digestion and LC-MS/MS analysis (results not shown) [64]. An 85% peptide coverage of the PH domain was obtained using MS/MS analysis (results not shown).
GST-GRP1PH binding specificity toward PI(3,4,5)P3 was analysed using both overlay phosphoinositide assay and Biosensor analysis (Figure 2). GST-GRP1PH was found to recognize specifically immobilized PI(3,4,5)P3 when compared to immobilized PI(4,5)P2 (Figure 2A) and 2B). A standard curve generated by plotting the average intensity of the dot blot versus the amount of immobilized PI(3,4,5)P3 showed that approximately 4ng (3.3pmoles) of PI(3,4,5)P3 could be detected in this assay.
Binding specificity was also characterized using biosensor analysis. GST-GRP1-PH was injected at various concentrations (1.5µM, 750nM, 375nM, 188nM, 94nM) over immobilized PC/PE/PI(4,5)P2 and PC/PE/PI(3,4,5)P3 liposomes (Figure 2C and 2D). GST-GRP1PH was observed to bind specifically to immobilized PC/PE/PI(3,4,5)P3 liposomes (Figure 2C) as compared to immobilized PC/PE/PI(4,5)P2 PC/PE (Figure 2D).
Kinetic constants were extracted from the biosensor curves from global analysis using 1/1 Langmuir binding with mass transfer. Analysis of the interaction between GST-GRP1PH and immobilized PI(3,4,5)P3 gave an apparent association rate (ka) of 4.5 x 104M-1s-1 and apparent dissociation rate (kd) of 1.6 x 10-3s-1 resulting in an equilibrium dissociation constant (KD) of 35.5nM.

Fluorescent imaging of GST-GRP1PH in resting and EGF stimulated HEK293T, LIM1215 and LIM2550 cells
Confocal microscopy was used to analyse PI(3,4,5)P3 localisation using GST-GRP1PH in resting and EGF stimulated HEK293T, LIM1215 and LIM2550 colonic carcinoma cells (Figure 3). EGF stimulation was performed for 1, 3, 5, 10 and 20 mins prior to cell fixation. Optimisation experiments indicated that 5 mins fixation in 4% formaldehyde was necessary and that the entire process of stimulation, staining and mounting should be completed within one day at room temperature. Unstimulated HEK293T cells showed a diffuse cytoplasmic and nuclear background staining (Figure 3). Increased fluorescence and localisation of GST-GRP1PH at the plasma membrane was observed 5 mins after EGF stimulation, with a concomitant reduction in cytoplasmic and nuclear staining (Figure 3). Membrane fluorescence and diffuse background staining returned to pre-stimulation levels 10 mins after stimulation. No background staining was observed when control experiments were performed with GST protein instead of GST-GRP1PH (results not shown).
Unstimulated LIM1215 cells also showed a diffuse cytoplasmic staining with evidence of some basal membrane staining (Figure 3). Increased fluorescence intensity and localisation of GST-GRP1PH at the plasma membrane, with concomitant decrease in background , were observed following 5 mins EGF stimulation. In these...

Find Another Essay On Characterisation of GST-GRP1PH

Psychological Egoism Theory Essay

2240 words - 9 pages The theory of psychological egoism is indeed plausible. The meaning of plausible in the context of this paper refers to the validity or the conceivability of the theory in question, to explain the nature and motivation of human behavior (Hinman, 2007). Human actions are motivated by the satisfaction obtained after completing a task that they are involved in. For example, Mother Teresa was satisfied by her benevolent actions and

How Celtic Folkore has Influenced My Family

1587 words - 6 pages Every family has a unique background that influences the way they live and interact with other people. My parents, who emigrated from Ireland to the States with my three brothers in 1989, brought over their own Celtic folklore and traditions that have helped shaped the way our family operates and lives. One aspect of folklore that has helped shape my family dynamic is the Celtic cross—both its background and what role it has played in our lives

Julia Margaret Cameron

1406 words - 6 pages At a time when women were looked upon as being homemakers, wives, mothers and such the late 1850's presented a change in pace for one woman in specific. Photography was discovered in 1826 and soon after the phenomenon of photography was being experimented with and in turn brought new and different ways of photo taking not only as documenting real time, but also conceptualizing a scene in which an image would be taken. Julia Margaret Cameron will

Evaluation of School Improvement

1403 words - 6 pages climate of the organization. Dr. Hunter indicated the learning environment should be positive, inviting, friendly, clean, and safe. Visitors should feel welcomed as they enter the school building. Dr. Hunter noted positive learning environment celebrates the diversity of the school population. The positive learning environment reflects the culture, belief, and values of the community, which is woven into the fabric of the school curriculum

Case Study: The Benefits of Animal Testing

1757 words - 7 pages Nine year old Amy has already had a rough start in life. She was born with an abnormal heart that hinders her everyday activities. Amy is unable to keep up with kids her own age because she often tires out easily. As a consequence, she has very little friends and is often alone. Amy is forced to take different medications everyday just to survive. Amy’s life consists of medicine, doctors, and constant hospital visits. However, Amy is due for a

Myth and Magic: Realism in "One Hundred Years of Solitude"

1531 words - 6 pages “He enjoyed his grandmother's unique way of telling stories. No matter how fantastic or improbable her statements, she always delivered them as if they were the irrefutable truth” (Wikipedia, 2011). Experiences are particular instances of one personally encountering or undergoing something and in these moments of time life changes for the best or the worst and memories are formed. These recollections such as riding your first bicycle, going to

Adiponectin: a Novel Indicator of Malnutrition and Inflammation in Hemodialysis Patients

2384 words - 10 pages Objective Protein-Energy malnutrition (PEM) and inflammation are common and overlapping conditions in hemodialysis patients which are associated with increased risk of morbidity and mortality. Adiponectin is an adipocytokine which is exclusively produced by adipose tissue. Few studies in hemodialysis patients have demonstrated that serum levels of adiponectin were significantly higher in malnourished patients compared to well-nourished ones. The

The Congo Free State: A Legacy of Apathy, Exploitation and Brutality

2298 words - 9 pages brutally exploited the inhabitants of the Congo, while at the same time robbed Congo of wealth, as the rubber was “sold” to the Europeans at prices far below what the rubber was actually worth. Only when others, specifically the British and the Americans, revealed the horrible conditions of the Congo, did the Belgian government reluctantly take over control of Congo, but the damage was done. Scholars, such as Martin Meredith, state that Leopold

Selective Exposition in The Lottery, by Shirley Jackson

1073 words - 4 pages Usually when someone hears the word “lottery” the first thing that comes to mind is a large sum of cash that people compete against highly impractical odds to win. Shirley Jackson’s story The Lottery might imply a similar conception based on the title alone, but the story is filled with unknowns never revealing exactly when and where the story takes place, or why the lottery exists; even what the lottery is isn’t revealed until the very end. Yet


1857 words - 7 pages INTRODUCTION I remember when I was a young child; I would always be scared whenever there was a severe storm outside that included thunder and lightning. This was especially true in the hours of darkness, when you could really see the lightning. As I grew older this so-called fear of lightning turned into a fascination for this weather phenomena. One of my most vivid memories of lightning as a young man was when I was flying to Florida, the

Maryland's Ecology and Environment

1130 words - 5 pages Maryland is the 42nd largest state, making it one of the smaller states in America. It is located in the South Atlantic region on the United States eastern seaboard. Prince George's is one of twenty four counties in Maryland. It is also the geographic center of the state. Maryland has a varied climate. The state is home to a variety of different ecosystems. This is also true of Maryland’s environment, which has the Atlantic Ocean

Similar Essays

When The Bubble Burst Essay

1539 words - 6 pages By the time I arrived state side from my second tour in the Middle East the housing bubble had already burst. I noticed a drastic change in the way that many of my friends and family were living. Several of my friends that worked in real estate had sold their boats and seconds houses. My own stock portfolio had lost a third of its value. My sister and her husband had defaulted on their home mortgage leaving them scrambling for a place to live. I

Phase Diagram Essay

4456 words - 18 pages Introduction: Chemical equilibrium is a crucial topic in Chemistry. To represent and model equilibrium, the thermodynamic concept of Free energy is usually used. For a multi-component system the Gibbs free energy is a function of Pressure, Temperature and quantity (mass, moles) of each component. If one of these parameters is changed, a state change to a more energetically favorable state will occur. This state has the lowest free energy

Revolutionary Work Of Art Essay

1890 words - 8 pages Walter Benjamin emphasizes in his essay, “The Work of Art in the Age of its Technological Reproducibility” that technology used to make an artwork has changed the way it was received, and its “aura”. Aura represents the originality and authenticity of a work of art that has not been reproduced. The Sistine Chapel in the Vatican is an example of a work that has been and truly a beacon of art. It has brought a benefit and enlightenment to the art

Enlightenment Thought In New Zealand Schools

1594 words - 6 pages In this essay I will be looking at how the political and intellectual ideas of the enlightenment have shaped New Zealand Education. I will also be discussing the perennial tension of local control versus central control of education, and how this has been affected by the political and intellectual ideas of the enlightenment. The enlightenment was an intellectual movement, which beginnings of were marked by the Glorious Revolution in Britain