What would have happened if we had adjusted the pH of our IDH extract to 4.5 and then loaded it onto the DEAE resin that was equilibrated in an IEC buffer also adjusted to pH 4.5? Explain
Isoelectric pH is a point at which the number of the negative charges in a protein are equal to the number of the positive charges. Therefore, the protein has not net charge and is therefore neutral. The protein has a net negative charge at a pH higher than the isoelectric pH and the protein has a net positive charge at a pH less than the isoelectric pH of the protein. In this experiment the isoelectric point of IDH protein is 8.08. The IEC (ion-exchange chromatography) buffer has pH=4.5 and the pH of the IDH is also adjusted to pH=4.5. As this pH is below the isoelectric point of this protein therefore the net charge on the protein will be positive. DEAE resin contains amino group and is thus positively charged. DEAE resin is an anion exchanger where the negatively charged proteins will bind and get eluted from the column based on the strength of the interaction between the positive charge on DEAE resin and the negative charge on the protein at a specific pH. If the pH of the IDH extract is 4.5 much below the isoelectric pH of this protein, the net charge of this protein will be positive so it will not bind to the DEAE resin which is also positively charged. In order to purify this protein the pH of the protein should be adjusted to a value more than the isoelectric pH so that the protein develops a negative overall charge and binds to the DEAE resin and can be eluted by washing it in the different salt gradient of KCl salt.
What would have happened if we had adjusted the pH of our IDH extract to 4.5...
SIRJE GEL OBJECTIVE: Partial purification of a protein, isocitrate dehydrogenase (IDH), using DEAE ion exchange chromatography. IDH will be eluted from the column using increasing concentrations of KCI. IDH will be tracked using the IDH activity assay and the extent of purification will be analyzed using SDS PAGE Last week we assayed for the activity of the enzyme, isocitrate dehydrogenase, from E.coli strains carrying a plasmid that over-expresses the E. coli IDH gene. To purify proteins from cel extracts, biochemists...
What would have happened to a calculation of the specific heat of your metal in the second trial if you had not carefully dried the metal before reuse? Clearly explain your reasoning. NG 3 3. What would have been the effect on your calculation of the specific heat of the metal had you forgotten to add the correction for your calorimeter constant? Explain. 4. How would your data have been affected if you had failed to dry your calorimeter between...
At one point or another we have all bumped our heads, stubbed our toes or had a mosquito bite. We all know the result which is swelling or inflammation. Using the medical terminology that you have learned this week discussed the following questions. What causes the accumulation of fluids in tissues and how does this fluid become lymph? 250 words
really har Opinion Shopping "We have had Paige & Gentry as our audi- tors for many years, haven't we, Jane? They have been here since I became president two years ago." Yes, Bob, I have been the CFO for seven years, and they were here before I came. Why do you ask?" Well, they were really tough on us "Well, ventry as our audi- really hard. You are a qualified accountant how can we handle this ecame president "Well, Bob,...
pH= 4.75 + log (5/5)= 4.75 pH= 4.75 + log (1/5)= 4.05 pH= 4.75 + log (1/10)= 3.75 pH= 4.75 + log 10= 5.75 pH= 4.75 + log 5= 5.44 Table 3: Sodium Acetate Data Sodium Acetate (g) Molarity of Sodium Acetate (Step 7) 4.0 g 0.4876 M Table 4: Buffer Solutions and pH Readings for Beakers A, B, C, D, and E Buffer mL of Acetic Acid mL of Sodium Acetate pH measured A 5 5 4.3 B 5...
We have just enough time to get our bearings when we see a huge enzyme (called hexokinase) closing in. We are wrapped into the active site of the enzyme. Your classmate sitting on Carbon #6 is brutally launched as the enzyme removes a phosphoryl group from ATP and attaches it to the carbon. We are released from the enzyme and the molecule on which we ride is now Glucose-6-phosphate. What is this type of active site binding called? What type...
A Routine Endoscopic Procedure Our mother usually had an endoscopic procedure every 2 years. We thought that she had too many visits with her gastroenterologist. She saw the gastroenterologist about every 30 or 60 days. When we asked the physician why our mother had to come in on a continuous basis, his response was “acid reflux.” She was taking a proton pump inhibitor for her acid reflux. Our mother had an endoscopy of her upper gastrointestinal tract in September 2006....
Most of us have had the experience at some point in our lives of sitting through a speech or oral presentation that was so dull we had to fight to keep our eyes open. And if we’re lucky, we’ve also experienced the opposite—presentations that really grabbed our attention and that stick in our memory long afterward. Find a link online to a speech or oral presentation that you think is memorable because it was either really good or really bad....
Explain what we would expect to happen to our sample variance if we change from sampling a population with 23 – 10 m2 plots to using 23 – 20 m2 plots? Be specific.
What happened to the spinach protein? Which layer would the proteins be in? Background needed: We ground up a spinach leaf in a mortar for 5 minutes with 3 mL of acetone and 3 mL of hexane. We then filtered the green solution (avoiding the ground leaves) into a filtering pipet. 2 mL of saturated aqueous sodium chloride were added to the test tube with the filtered solution in order to make **two layers**. The top, more green layer was...