PROTEIN ISOLATION and QUANTITATION

Protein Isolation

For this and the other steps, the sample is required to be transport at appropriate conditon to get rid of protein denaturation. In addition to that all information about the transportation have to be recorded.

Protein purification involves many steps. We can isolate proteins from various type of samples such as biopsy, urine or blood. The purification steps and materials can vary according to type of sample and the desired part of protein complex and optimization steps can be performed by our unit.

Purification steps can be divided into two main parts that are mechanical and the chemical application.

If the samples has rigid property; it can be freezed using liquid nitrogen and then the sample is crushed into small pieces.

After this step; the sample is incubated with lysis buffer to purify protein from other complex. At this step for homogenization purpose sample can be sonicated with SONICS Vibra Cell.  At this step sample volume is maximized because of lysis buffer treatment. The sample is then placed in appropriate centrifuge for the precipitation step.

Centrifugation is a process that uses centrifugal force which can be used for the separation purpose. By using this force it is possible to get fraction in a mixture depending on their densities and mass. In our unit we use  OPTIMATM L-100 XP (Beckman Coulter) to purify protein at high efficiency.

After this purification step, it possible to concentrate protein mixture by using different steps such as ultrafiltration or  lyophilization steps. In our unit there are diiferent type of ultrafiltration tubes that are able to separate proteins of different sizes. In addition to that, by using MILLROCK TECNOLOGY Lyophilization, proteins can be concentrated.

Protein Quantitation

Generally spectrophotometric measurement is used for the protein concentration calculation. There are several options for this measurement and in our unit we perform Bradford, BCA and Lowry Methods which are known as colorimetric assay and for the spectrophotometric measurement Perkin Almer Victor 3 V Multilabel Counter is used.

a-Bradford Method

In this method Coomassie dye is used and this logic behind this measurement is binding of Coomassie Dye ( Brillant Blue G250) binding to aromatic amino acids and measurement is performed at 595 nm. In our unit BSA is used as standards calculation and based on standart curve sample concentrations can be calculated.  Compare to other methods, this method is fast and more sensitive.

b-Bis-Cinchinonic Acid (BCA) Method

Spectrophotometric assay based on the alkaline reduction of the cupric ion into the cuprous ion by interaction with protein at 562nm.

c-Lowry Method

In this protein quantitation method, the logic is the binding of copper ions with the protein peptide bonds under alkaline condition, followed by the oxidation of aromatic proteins. This method is also highly sensitive in protein detection.