5. Affinity Chromatography.

Affinity chromatography was originally developed for the enzyme purification. Now, it has been extended to immunoglobulins, membrane receptors, nucleic acids, nucleotides, whole cells and cell fragments.

It is theoretically capable of giving absolute purification from complex mixtures in a single process.

Affinity precipitation, in which the ligand is attached to a soluble carrier that can be subsequently precipitated by for example pH change.

Affinity Partition, in which the ligand is attached to a water-soluble polymer such as polyethylene glycol and which, with the ligand bound, partitions into an aqueous polymer phase that is in equilibrium with a pure aqueous phase.

In this chromatography, the most commonly used matrix are, cellulose, agarose, polyacrylamide, polystyrene, cross-linked dextrans and porous glass antisilica.

Applications of Affinity Chromatography:

1. used to purify a large variety of macromolecule such as enzymes, membrane receptors, nucleic acids.

2. Immobilized enzymes are purified.

3. mRNA can be isolated.

4. Metal chelate affinity chromatography is the logical extension technique.

5. proteins can be separated based on their molecular weight.

6. Papain and Urease can be separated.

7. It has an application in separating DNA and RNA

Comments

Statistics in Flow Cytometry Data and "MFI" values

The speed of the flow cytometry offers wide range of data points and data plots. Due to its sensitivity and versatility, it has been used extensively. Flow cytometer can detect up to 1000s of cells per second.. But, it should be noted that the flow cytometry also involves the statistics, its significance, calculating the fluorescence intensity.. Statistics in flow cytometry involves, total number of data points acquired, percentage population, most importantly mean and median fluorescence intensity, and others. Most of us get confused about the term "MFI", whether it means mean fluorescence intensity or median fluorescence intensity.. Truth is, both.. It means it can be described as either mean or median fluorescence intensity. Some researchers use Mean for MFI and some use Median for MFI values. (Keep...

Protocol: Sample preparation for Cell Cycle Analysis by Flow Cytometry

Protocol: Preparation of Samples for Cell Cycle Analysis by Flow Cytometry: 1) Suspend approximately million cells (10^6) in 1mL of phosphate-buffered saline solution (1x). And with the help of pipette, gently aspirate about 7-8 times, to make the single cell suspension. 2) Take a centrifuge tube and add 4mL of 70% ethanol. (70% ethanol should be one ice before using). and add 1mL of cells in PBS to this tube. Keep the tubes on ice for minimum 2 hours at 4℃.. This step will fix the cells. ( cells can kept at 4 ℃ for a week). 3) Centrifuge the ethanol fixed cells for 5 minutes at 300g. Carefully take out the supernatant. Do not disturb the pellet. 4) Add 5mL of cold 1x PBS to this pellet and suspend the cells using pipette. 5) Centrifuge the tubes at 300g for 5 minutes. and discard the supernatant. Again do not disturb the pellet. 6) if required, step 5 can be repeated for one more time. 7) Add 1mL of Propidium Iodide (PI...

Fluorescence Spectrum Viewers Tool.

Spectra Viewers are the fluorochrome tools which help in understanding the wavelength ranges of excitation and emission of fluorochromes.. These tools are very helpful in determining specific laser, detectors and range of fluorescence spectra. When Multi-Color Flow Cytometry is the experiment, then these must be used to analyze emission spectrum, fluorescence spill over and compensation values. How these tools help.. First, Lets consider a simple example of analyzing the spectrum for Green Fluorescence Protein (GFP) and you want to know the excitation wavelength and emission wavelength range for GFP. By knowledge, you know that GFP will excite with 488nm Blue laser and emits the green fluorescence at apporximately 510nm.. Now, just open a spectrum viewer and select the GFP as shown below. Now, you have analyzed GFP fluorescence and received the knowledge that It excites with 488nm Blue Laser and emission maximum is...

BIOMAG

Search This Blog