Protein Purification Using Affinity Chromatography

Protein Purification Using Affinity Chromatography Protein Purification Using Affinity Chromatography ABSTRACT: The principle behind this lab experiment was to purify the His-tag protein RNase H by implementing a technique called affinity chromatography. This technique is unique in its purification technological apparatus because it allows the purification of a biomolecule in accordance to its individual chemical disposition. A mini-column is prepared using 0.5 mL of Ni-NTA agarose and washed with 10 mL of DI water. To begin the purification process, a sample of E.coli containing His-tag H is ran through a series of buffers to wash and remove unbound proteins, and then eluted to obtain the desired protein. During this procedure, the original flow through, eluting buffer flow through, washing buffer flow through and the unused eluting buffer will be retained and stored for the latter experiment. INTRODUCTION: Affinity chromatography is a technique for the purification of proteins. It isolates the transcription factors and purifys proteins by binding to a specific DNA sequence. The solution is passed down a column that contains the DNA sequence attached within the matrix. The proteins containing a relatively high affinity for the specific sequence are gravitated towards the matrix where it will remain and bind to the sequence. As given by the name itself, affinity chromatography is highly selective henceforth, superior resolutions and extreme capacity for proteins in query. Affinity chromatography isolates the proteins by means of a rescindable interaction linking the protein or in some cases a group of proteins, and a distinctive ligand attached to a chromatographic medium. Affinity chromatography is an efficacious method when the interactions between the protein and the molecule of interest is highly specific. However, the purification process can be a tad tedious and time-consuming. So to expedite the recovery of proteins while efficiently purifying recombinant proteins, affinity tags are introduced to various methods. The majority of the affinity tags are grouped as either a peptide or protein, which selectively adheres to the immobilized metal ion on the affinity column. The introduction of the affinity tags allows us to purify the proteins using affinity chromatography by taking advantage of the interaction associated with the metal ions and the protein molecules. The affinity tag is the amino acid Histidine, called the His-tag. The tagged proteins are passed through the column of beads containing covalently attached, immobilized nickel (II) or other metal ions (Biochemistry, 2015). Histidine is known to display the greatest interaction with the immobilized transition metals, such as Ni2+, therefore, they are the most commonly used affinity tag. This is due to the ionization property of the amino acid residue. Histidine contains an imidazole ring, that can bind and release protons depending on the surrounding environment of the matrix (Biochemistry, 2015). In this experiment, the matrix used for purifying the protein containing the His-tag is the Ni-NTA Agarose. The His-tag binds to the immobilized nickel (II) with great affinity and specificity, while the other proteins molecules are weakly bonded or end up getting washed out during the washing step. The E.coli lysate is what is loaded into the minicolumn affinity matrix. The bound proteins remain attached while the other proteins wash through the matrix. After several washes, the bound His-tag protein is eluted from the column using an eluting buffer which will decrease the binding affinity and displaces the protein. The His-tag protein can also be eluted with imidazole, which is known to be the most generally used elution agent. In this experiment, the protein was purified by collecting the supernatant from each wash series which ran through a Ni-NTA affinity column. Each buffer contained different concentration values of Imidazole in increasing order, starting with 5 mM, 20 mM, and ending with 250 mM for the eluting buffer. The final flow through of eluting buffer wash contained the completed purified protein. EXPERIMENTAL PROCEDURES: MATERIALS: 0.5 mL of E.coli lysate containing over-expressed His-tag RNase H 5 mL of Loading Buffer: 20 mM Tris-HCl, 0.5 M NaCl, 5 mM Imidazole, 10 % Glycerol 2.5 mL of Washing Buffer (2xs): 20 mM Tris-HCl, 0.5 M NaCl, 20 mM Imidazole, 10 % Glycerol 1 mL of Eluting Buffer: 20 mM Tris-HCl, 0.5 M NaCl, 250 mM Imidazole, 10 % Glycerol 10 mL of Glycerol (3xs) 0.5 mL Ni-NTA Agarose DI water HCl pH meter Mini-column PROCEDURE: Prepare the buffer solutions using the calculated values and adjust the pH with HCl until you reached a pH ofà ¢Ã¢â€š ¬Ã‚ ¦, and then top off to 100 mL with water. **NOTE: The glycerol, Tris, NaCl, and imidazole can be added to 80 mL of water and the volume topped off to a total of 100 mL after the pH adjustment. **Please refer to the data table for the appropriate values. To prepare the minicolumn, add 0.5 mL of Ni-NTA Agarose to the minicolumn and wash with 10 mL of DI water. Once the water has flowed through the column, add 5 mL of the loading buffer. When the loading buffer has gone through, proceed and add 5.0 mL of the E.coli lysate and save the flow through for the following lab. Using the prepared washing buffer, wash the minicolumn twice with 2.5 mL of the washing buffer and retain the flow through from the first wash only for the latter experiment. Once the washing buffer has completely flowed through the column, wash the minicolumn with 1 mL of the eluting buffer, and again save the flow through. The flow through from the eluting buffer was contains the final purified protein. Also, save 15 mL of the unused eluting buffer for the following lab experiment. Place all the saved flow through in the appropriate storing tube and label accordingly, including the initials of each group member or a distinctive marking so that it can be easily reclaim ed in the next lab. Hand the labeled tubes over to the TA for proper storing, you should have a total of four solutions. DATA TABLE: To make 100 mL of Loading Buffer 100 mL of a 10% Glycerol Solution 20 mM Buffer 5 mM Solution 0.5 M Solution Tris Needed Imidazole Needed NaCl Needed Glycerol Needed Loading Buffer 0.242 g 0.3404 g 2.922 g 10 mL To make 100 mL of Washing Buffer 100 mL of a 10% Glycerol Solution 20 mM Buffer 20 mM Solution 0.5 M Solution Tris Imidazole NaCl Glycerol Washing Buffer 0.242 g 0.1362 g 2.922 g 10 mL To make 50 mL of Eluting Buffer 100 mL of a 10% Glycerol Solution 20 mM Buffer 250 mM Solution 0.5 M Solution Tris Imidazole NaCl Glycerol Eluting Buffer 0.121 g 0.851 g 1.461 g 10 mL REFERENCES: J. M. Berg, J. L. Tymoczko, G. J. Gatto, Jr., L. Stryer, Biochemistry (8th ed., pp. 70-71). W.H. Freeman Company. Hengen, P. N. (1995). Purification of His-Tag Fusion Proteins from E.coli. Trends in Biochemical Sciences, 20(7), 285-286. https://www.qiagen.com/us/shop/sample-technologies/protein/expression-purification-detection/ni-nta-agarose/#orderinginformation Biological Chemistry Laboratory Manual, (2017).

Middle School Essay

So I am not really ready for middle school and btw my name is Aniah Stitt and I am leaving the 5th grade from Reedy Creek Elementary. I am really scared because I was supposed to go to a school called Northridge Middle and that is a really bad school! People say they have bad kids and that is true but every school has bad kids and all of my friends are going there and I have no friends from my school going to my new school(well my friend Sharifa, Mackenzie, and this boy named Justin)and its really hard. The thing is I have friends in the 7th, and 8th but I  won’t be with them. I know what I am wearing but I don’t have my whole day planed out and do we have to wear book bags and btw we don’t get lockers till 8th grade and it has to be a privilege! And I forgot to tell you I am going to Randolph Middle and it will be so hard because it’s an IB school and I decided to write about my junior high life. Welcome!!!!! and it will be a long year I can already tell. Ok, first things first I am in Ms. Pfahler’s Homeroom and my math teacher is Mr. Dunn and Science and Social Studies is Mrs. Mitchell. People say I have the good teachers because I am in the building but I say I also have the boring ones, but I officially don’t know yet, so I guess I have to wait and find out. My school starts at †¦Ã¢â‚¬ ¦. And ends at †¦Ã¢â‚¬ ¦.! We also have to do community service for 10 hrs! but I did 36 because of operation charlotte at Hickory Grove Baptist Church (HGBC) and I like basically do everything there and they have a school that I wanted to go to HGCBS (hickory grove baptist) well get comfortable for a year of sixth grade at Randolph Middle!

Is Technology Destroying Our Life? Essay

Technology is a wonderful thing giving us almost instant access to the world’s information. It makes our life easier, and enables us to stay in touch with distant friends and family. But it’s not all great. From big stuff like cancer to small stuff like being distracted texting, technology is killing us slowly every day. Texting while driving or walking is a killer People hate talking to people on the phone, so they’ve taken to texting. The problem with texting You need to have your eye on your phone to make sure you get the message right. Fine when you’re sitting still, a disaster when you’re driving. From 2001 to 2007, 16,000 people died from texting while driving. Even out of the car, texting is a disaster. Emergency rooms were seeing over 1,000 visitors who were getting into accidents because they were distracted trying to walk and text. We sit around using computers at work Sitting all day long is terrible for you. It makes you fat and weak, and can actually increase the odds of you dying sooner than later. So, what’s that got to do with technology? Well, thanks to technology we’re sitting at work more and more using computers to get our jobs done. Cell phones are probably giving us cancer This is one that remains inconclusive and divisive. Researchers haven’t found a definitive link between cancer and cell phones, yet there is a body of evidence linking the two. If you have a cell phone constantly glued to your ear, there is at least some reason to be worried about a tumor forming. Just use a headset to play it safe. Facebook is fueling divorce, which can lead to depression Facebook is actually leading to divorce for married couples. Spouses can get jealous when they look at who is friends with who, or if they see flirtatious messages being sent. It drives a wedge in the marriage, which can end in divorce. Divorce can lead to depression, which can lead to suicide. Craigslist, if you’re not careful, can be very deadly Craigslist is an amazing service making it much easier for us to sell old crap with minimal hassle, or find a new apartment without a broker. However! If you’re not careful with how you approach Craigslist, you could get yourself in hot water. Take a look at Congressman Chris Lee, who used Craigslist to look for hook ups. It ruined his career (and probably his marriage too.) There are also more serious examples of danger from Craigslist like Philip Markoff, the so-called Craigslist killer. Cellphones on planes can clog up the radio signals There’s something about being on a plane that when the crew tells you to turn off your phone you can’t help but scoff and try to squeeze in reading a few more tweets, or checking email one last time. What’s the harm in it? Well, the FCC worries that a plane load of people doing just that can scramble radio signals and cause problems for the plane. People are losing their sleep thanks to TVs, cellphones, and other screens Staring at bright screens before you go to sleep can disrupt your sleep, says a Berkeley sleep specialist. The bright screens of an iPhone, iPad, or even TV, disrupt your body’s natural rhythms at night. If you don’t get enough sleep, your lifespan can be significantly shorter according to research from the University of Warwick. Electric cars are dangerous for the blind Electric cars and hybrids are silent, and as such, they are a threat for people that rely on hearing cars coming. Hybrid and electric car makers know that silent cars are a threat, and they’re considering adding noise makers to the car to alert the outside world. More efficient farming and shipping is making us fat Our instinct as human animals to eat fatty and sugary things — things that give us the most energy to store because throughout most of evolution we did have to go through stints of deprivation. In modern life, we still have the same tastes but we don’t need to anymore. Now, thanks to technological innovations in farming and shipping, fatty and sugary things are available everywhere—and super cheap. This is making us fat, and being fat is killing us (and costing us lots of money). Finally I hope to forget the technology a little bit and take care about each other more because our social life is going to die . I think if we stay like this we will lose our friends , maybe our family and our humanity.

True Story of the Lacks Family - Free Essay Example

Sample details Pages: 2 Words: 541 Downloads: 5 Date added: 2019/08/07 Category Medicine Essay Level High school Topics: Henrietta Lacks Essay Did you like this example? A young African American woman named   Henrietta Lacks was born and raised in Virginia in the 20th century. She was mother to 5 children and had lived in poverty. She had a brief life due to having cervical cancer which is the cancer of the cells in the cervix. Don’t waste time! Our writers will create an original "True Story of the Lacks Family" essay for you Create order She had lots of struggles throughout her life, despite all that she worked hard for her family and lived her life to the fullest. Her unfortunate illness, has helped millions of people. Rebecca Skloot, a young woman who came across Henrietta Lacks in class, was intrigued with her. She wanted to right a book about her, whats a better source than Henriettas family themself. Beginning with her daughter, Deborah Lacks. The book that was published in 2010 and an article titled, Henrietta Lacks Immortal Cells   written by Sarah Zielinski, in where she discussed the   morally correct issues on how the Lacks werent pleased about how doctors had taken advantage of their mothers cells and had used them as research without permission. Sometimes you have to make decisions for the greater good and sometimes it doesnt make everyone happy, like in this case.   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   The hospital that had used the cells without permission was John Hopkins Hospital, located in Baltimore, Maryland. Doctors and scientists would avoid using Henriettas real name by calling them HeLa cells according to the book, The Immortal Life of Henrietta Lacks. The family was also intensely involved in the research, with doctors using the familys genes without the consent of the family. In the book it states, the last thing he remembered before falling unconscious under the anesthesia was a doctor saying his mothers cells were one of the most important things that had ever happened to medicine. Sonny woke up in more than $125,000 in debt because he didnt have health insurance to cover the surgery. The family had made no money in the discoveries. The family had financial struggles. According to the article that I had mentioned before, it took almost a year even to convince Deborah to talk to Rebecca because she knew Rebecca was desperate to learn about her.   Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   HeLa cells were such an astounding use to medicine. HeLa has helped doctors and scientists with many things. According to the article, scientists had sent the cells to space to see how they would react and adapt in zero gravity. During the space mission, a chemical was accidentally spilled on a cell and spread out its tangled chromosomes, in which we discovered, instead of 48 chromosomes, which we initially thought was right, there was instead 46 chromosomes with 23 pairs. Doctors also used it to test the polio vaccine.  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚     Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚  Ã‚   Both the book and article deliberate about the ethical and morally correct conflicts on how the Lacks were upset on how their family was taken advantage of, regarding the usage of their genes and the cells of their late mother, without permission. Its kind of up for debate, because many people think if Henrietta was alive she wouldnt mind the hospital using her cells for research purposes. HBO has turned this story into a movie based on the book   The Immortal Life of Henrietta Lacks   that premiered on April 22nd, 2017. It told how Rebecca Skloot had found the true story of the Lacks family.