what is blood
Blood is a special type of connective tissue in which the extracellular matrix (plasma) is fluid. In other forms of connective tissue such as cartilage, tendon, and bone, the matrix is fibrous or solid in composition. Red Blood cells (RBCs), also known as erythrocytes, respond to different concentrations of saline solutions as a result of osmosis.
How do RBC’s react to isotonic, hypertonic, and hypotonic environments
RBC’s in isotonic saline have a net zero exchange of water across the membrane.
RBC’s in hypertonic environments have a net loss of water resulting in crenation
RBC’s exposed to a hypotonic environment will have a net influx of water causing the cells to lyse (burst open)
what is Fractionation
Fractionation is the procedure by which cells or tissues are broken down, and their components separated and isolated so they can be analyzed. In addition to the disruption of tissues and or cells, fractionation also involves differential centrifugation to separate major kinds of components.
what is the purpose of fractionation
The ultimate purpose of a fractionation dictates the volume of sample that should be used. You would need a very large volume, for example, if you wished to purify a specific protein that occurs in very low concentrations.
what can blood be separated into
Blood can be separated by osmotic lysis and differential centrifugation to produce three protein-containing fractions: plasm, Lysate (red blood cell cytoplasm), and cell membrane.
how do you separated a blood sample
A blood samples can be centrifuged at a low speed to separate the plasma from the erythrocytes. The erythrocytes can then be lysed by placing the cells in a hypotonic solution causing them to burst. Further centrifugation then allows for isolation of the lysate and cell membrane.
Why do proteins degrade
Proteins easily degrade when handled in a laboratory setting due to ubiquitous cellular proteases released from cellular compartments following osmotic lysis.
what are the two ways to prevent protein degradation
Important handling procedures can help to minimize the degradation of proteins in lab samples such as:
- Keep samples in ice (cost effective)
- Use protease inhibitors (expensive, best for sensitive research)
By keeping your samples on ice enzymatic degradation by proteases is slowed. These enzymes are found ubiquitously within all cells. There are many types of proteases that can targeted by protease inhibitors to prevent sample degradation.
how do you store protein samples
Protein samples are stored at ~ -20 ºC to -80 ºC often in the presence of glycerol or other viscous liquids. This allows enzymes to remain active following thawing. Proteins cannot usually withstand several freeze thaw cycles and so are aliquoted in small volumes prior to being frozen.
what dye did we use in lab 6
For Lab 6 we used the acidified component Coomassie Brilliant Blue dye, which reactions and binds quantitatively to arginine and to aromatic amino acids such as tyrosine, phenylalanine, and tryptophan residues in the proteins.
how can you measure the protein concentration of a sample
The protein concentrations of a sample can be measured during the Bradford assay.
what happens to the bound dye
The bound dye undergoes a shift in wavelength from 470 nm to 595 nm (colour blue). Which can be measured from a spectrophotometer. It is a highly specific and very sensitive, and the dye-protein complex formed remains stable for about an hour.
how do you translate the absorbance into protein concentration
In order to translate the absorbance into protein concentration, a series of known concentrations of a protein, called standards, are tested with Bradford reagent to prepare a standard curve.
what did we use in lab 6 to make the standard curve
In lab 6 we used Bovine serum albumin (BSA) which is extracted from the blood of the cattle. Any other protein containing sufficient arginine and aromatic amino acids could have been used.
what is the protein components of plasma
The protein components of plasma consist mainly of serum albumin, globulins, clotting and hormone-bonding proteins.
what is the lysate fraction made of
The lysata fraction consists mostly of the protein hemoglobin from the erythrocyte cytoplasm.
what does the membrane fraction consist of
The membrane fraction consists mainly of integral membrane proteins (=integrins), glycoproteins, and cytoskeleton proteins.
what do we assume in lab 6, related to hazards
Animal products provided by commercial sources are subject to regulatory controls to ensure they are safe and uncontaminated. In spite of these precautions, it is normal to assume that all animal products are potentially hazardous.
what is fibrinogen
In humans fibrinogen, an important protein for blood clotting, is present in concentrations of less than 10 ug/mL
what should the protein concentrations in blood be for humans
For human blood the adjusted protein concentration should be roughly:
4-10 mg/mL for the Plasma fraction, averages 7
20-50 mg/mL for the Lysate fraction, averages 35
1-8 mg/mL for the membrane fraction, averages 5
