Cell Bio Lab #4 - Protein Isolation and Estimation - Review

Physical rupture and chemical lysis

What are the two general methods available to lyse cells?

Physical rupture

This method of lysing cells includes mechanical disruption, liquid homogenization, sonification, freeze/thaw, and manual grinding

Chemical lysis

This method of lysing cells uses detergents like sodium dodecyl sulfate (SDS), Triton X-100, NP40, Tween 20, and osmotic agents such as mannitol (where a high mannitol concentration ruptures membranes)

- Variable yields

- Variable viscosities

- Expensive instruments

- Subsequent heating of cell sample

What are the disadvantages of lysing cells through physical rupture?

It is very effective, however, there are many disadvantages

What is an advantage of lysing cells through physical rupture?

- More reliable than physical rupture

- Rapid process

- Reproducible results

- Results in a high protein yield

What are the advantages of lysing cells through chemical lysis?

Some methods interfere with the salts used in lysis buffers

What is a disadvantage of lysing cells through chemical lysis?

SDS, Triton X-100, NP40, Tween 20, and osmotic agents such as mannitol

What mild detergents are used in chemical lysis?

Mechanical disruption

This method of physical rupture was used in lab in the form of scraping. The disadvantage of this is that you can produce variable yields

Liquid homogenization

This method of physical rupture is similar to using a blender to destroy cell membranes. A disadvantage of this is variation in viscosity

Sonification

This method of physical rupture uses high frequency sounds to break apart cells. A disadvantage of this is that the instruments required are very expensive

Freeze/thaw

This method of physical rupture involves flash freezing and flash thawing to crystallize the cytoplasm and rupture cell membranes. A disadvantage to this is the repeated heating of the cell sample

Home detergents or soaps are very concentrated and denature proteins. They are potentially impure for the purpose of a lysis buffer, and the concentration of them is very high, so it instantly destroys the cell membrane and denatures proteins. Protease inhibitors are not likely to prevent denaturation caused by home detergents or soaps

Why should we not use home detergents or soaps to make the lysis buffer?

Protease inhibitor is added to deactivate protease so that it does not dissolve/denature proteins when they enter the solution after cell membranes are disrupted/lysed

What is the purpose of using protease inhibitor in lysis buffer cockatil?

y=0.00029x+0.0332

0.0665=0.00029x+0.0332

0.0333=0.00029x

x=113.83 ug/ml

The standard curve equation for protein estimation was determined to be y=0.00029x+0.0332. Calculate the concentration of protein (ug/ml) present in the sample with an OD value (at 562nm) of 0.0665, as determined using BCA protein assay kit.

Cu2+ in the reagent is converted (reduced) into Cu1+ in the presence of proteins and in an alkaline medium. The Cu1+ ion forms a complex with two BCA molecules which turns a purple color that appears red at an absorbance of 562nm.

BCA Protein Assay is a detergent-compatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein. Cu2+ is reduced to Cu1+ by protein in an alkaline medium, and the cuprous cation (Cu1+) is is very sensitive and selective to colorimetric detection when combined with two BCA molecules. The purple-colored reaction product is formed by the chelation of two BCA molecules and one cuprous ion. This water-soluble complex exhibits a strong absorbance at 562nm. The BCA method is not a true end-point as the final color continues to develop. However, following incubation, the rate of continued color development is sufficiently slow to allow large numbers of samples to be assayed together. The macromolecular structure of protein, the number of peptide bonds, and the presence of four amino acids (cysteine, cytosine(?), tryptophan, and tyrosine) are responsible for color formation with BCA. Protein concentrations are determined and reported with reference to standards of a common protein such as bovine serum albumin (BSA)

State the principle of BCA protein assay

To rupture the cell membrane in order to release intracellular proteins

What is the purpose of cell lysis?

This limits denaturation of the proteins by slowing down molecular movement.

What is the purpose of placing the protein samples in ice throughout the experiment?

562 nm

At what wavelength does the Cu1+ ion + 2 BCA molecule complex exhibit a strong absorbance? (appears red)

Mechanical destruction and the use of SDS

What combination of physical and chemical lysis did we use in lab?

Supernatant

After mechanical destruction and the use of SDS, the debris contains cell membranes and insoluble materials. Between the supernatant and the pellet, where are the whole cell proteins present? This is the component that needs to be transferred

Bovine serum albumin (BSA)

Protein concentrations are generally determined and reported with reference to standards of a common protein such as what?

Bicinchoninic acid (BCA)

What compound does BCA stand for?