The Principle Of Affinity Chromatography

Salting out is the first isolation technique carried out for this lab. This isolation technique separates the Rubisco based on solubility. The method of salting out requires an addition of salt such as ammonium sulfate to the solution containing protein Rubisco to allow precipitation (Duong-Ly & Gabelli, 2014). The additional of ammonium sulfate to the Rubisco

Again, label 7 1.5ml tubes 0 thru 6. Place 15μl of each serially diluted extract into its corresponding labeled tube. Next add 465μl of media into each tube. Then 60μl of Alamar blue in each tube. Finally add an additional 60μl of cells (adjusted to 10,000 cells/20 μl). Vortex each tube for 5 seconds. Now, take 3 different samples 190μl samples of concentration 0 and put it in Wells A2, B2, and C2. Repeat this step again by taking 3 more different 190μl samples of concentration 1 and putting it in wells A3, B3, C3. It should be noted that it is important to vortex each 1.5μl tube again be-fore putting it into the 96 well plate. Contin-ue this same procedure consecutively for the re-maining concentrations.

tralization solution and centrifuging it for 5 minutes. The Wizard miniprep kit is used to bind the

1) Pour 25 mL of the 1 M hydrochloric acid into the beaker and rinse the solid by swirling the acid around in the bottom of the beaker.

If there are any lab’s instructions that I should know, please describe it to me. I am afraid in making any mistake again.

3. Use a sterile pipette to transfer 0.1 ml of each dilution on to a MacConkey agar plate.

Number three test tubes 1 to 3. Add 2ml of monosaccharide solution to tube 1. Rinse out afterward in a cup of water.

The agar plates will be divided into 3 sections (one for each filter paper discs) using any form or marker. Using sterilized forceps, put the filter paper disc at the center of each area in an agar plate. Apply 20µl of each concentration for 10 agar plates. This includes distilled water (negative control), 100% ethanol and the common antibiotic. Observe for the zone of inhibition within 16 hours and above (Tendencia, 2004). To measure the zones of inhibition, use a ruler and record the diameter in terms of millimeters.

Preparing the Column: Add a small amount of cotton into the column covered with a thin layer of sand and clamp it. Dissolve silica gel in eluent and add it to the column while draining the eluent to make the stationary phase. Add another layer of sand to keep the silica gel in place. Make sure there are always enough eluent to keep silica from dry. The column is ready to use once the eluent levels with the top sand layer.

Affinity chromatography can also be used to isolate specific antibodies or antigens from antiserum. Antigen is covalently bound to small, chemically non-reactive beads and loaded onto a column. Antiserum is passed over the beads which results in binding of antibodies to the antigen. Specific antibodies are then eluted by normally lowering the pH to 2.5 or raising it to >11. Similarly antigens can be purified by binding specific antibodies to the

The initial motivation for scientists to investigate more about dye ligand affinity chromatography was given after the interactions that took place between Blue Dextran, a Cibaron Blue and dextran conjugate, which is used as a void marker in size-exclusion chromatography, and particular kinases. Until then, only purification of various proteins by size-exclusion chromatography with Blue Dextran, like for example, erythrocyte pyruvate kinase, phosphofructokinase, glutathione reductase,

9. Use a sterile transfer pipette to add 100 μl of cell suspension from the “+” culture tube onto your LB/Amp/ara and LB/Amp plates. Then, using a new sterile transfer pipette, add 100 μl of cell suspension from the “-“ culture tube onto your LB/Amp and LB plates. Remember to only open the lid enough to get the tip of the pipette inside to deposit the 100 μl to prevent possible contamination of the culture by bacteria in the air.

The first included Cibacron Blue Sepharose CL-6B, proteins, and egg white lysozyme. They tested the proteins and egg whites binding ability to the Cibacron Blue. To make matters easier, they only searched for absorption of pure proteins from 0.05 M Tris-HCl buffer, pH 7.2. This was achieved by eluting the absorbed proteins with 1M KCl In in 0.05 M Tris-HCl buffer. The second type included immobilized monoclonal antibodies and the absorption of E. Coli B-galactosidase against B-galactosidase. This was chosen due to the simplicity of measuring the activity of B-galactosidase that could then tell us the concentration of the antigen. The only downside to this approach was B-galactosidase has a much higher molecular weight that most antigens commonly used by this method. This means the large molecule will have trouble penetrating the pores in the immuno-absorbents. This was done by immobilizing the antibodies on a silica-based adsorbent, which was then added to PBS buffer. The buffer contained antibodies. The flask they were in was then shaken for four hours, followed by removal of all the unreacted protein that was done by using a funnel with sodium chloride solution. To wash the gel they used propionic acid and then stored the adsorbent in PBS buffer. Finally to elute the absorbed B-galactosidase, they used 6 M urea in 20 mM Tris-HCl, 10 mM ethylene diamine tetraacetic acid, and 10 mM sodium Chloride, 0.1 M 2-mercaptoethanol, pH 7.2 (Chase

The gel will polymerize at room temperature in 20-40 mins. Pour electrophoresis buffer on the gel and remove comb. Pour

In the preparation of agarose gel, agarose powder will be mixed with buffer. Agarose powders need to be weighed first before mixing it with buffer. When 1 gram agarose gel is added, it will be followed by 100 ml of buffer. The ratio is 1:100 for agarose powder and buffer respectively. Then, the solution of agarose powder and buffer is put in the microwave in order to melt the agarose until the solution become clear (Carson & Robertson, 2005).