Experiment: Bradford protein assay give the answers (4-5 lines) of review questions in the end. answer should be logical and understandable and without plagiarism. avoid copy pasting. Background information: The Bradford assay is very fast and uses about the same amount of protein as the Lowry assay. It is fairly accurate and samples that are out of range can be retested within minutes. The Bradford is recommended for general use, especially for determining protein content of cell fractions and assessing protein concentrations for gel electrophoresis. The method described below is for a 100 µl sample volume using 5 ml color reagent. It is sensitive to about 5 to 200 micrograms protein, depending on the dye quality. In assays using 5 ml color reagent prepared in lab, the sensitive range is closer to 5 to 100 µg protein. Scale down the volume for the "micro assay procedure," which uses 1 ml cuvettes. Protocols, including use of microliter plates are described in the flyer that comes with the Bio-Rad kit. Principle: The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. The assay is useful since the extinction coefficient of a dye-albumin complex solution is constant over a 10-fold concentration range. Equipment: In addition to standard liquid handling supplies a visible light spectrophotometer is needed, with maximum transmission in the region of 595 nm, on the border of the visible spectrum (no special lamp or filter usually needed). Glass or polystyrene (cheap) cuvettes may be used, however the color reagent stains both. Disposable cuvettes are recommended. Procedure: Reagents 1. Bradford reagent: Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol, add 100 ml 85% (w/v) phosphoric acid. Dilute to 1 liter when the dye has completely dissolved, and filter through Whatman #1 paper just before use. 2. (Optional) 1 M NaOH (to be used if samples are not readily soluble in the color reagent). The Bradford reagent should be a light brown in color. Filtration may have to be repeated to rid the reagent of blue components. The Bio-Rad concentrate is expensive, but the lots of dye used have apparently been screened for maximum effectiveness. "Homemade" reagent works quite well but is usually not as sensitive as the Bio-Rad product. Assay: 1. Warm up the spectrophotometer before use. 2. Dilute unknowns if necessary to obtain between 5 and 100 µg protein in at least one assay tube containing 100 µl sample 3. If desired, add an equal volume of 1 M NaOH to each sample and vortex (see Comments below). Add NaOH to standards as well if this option is used. 4. Prepare standards containing a range of 5 to 100 micrograms protein (albumin or gamma globulin are recommended) in 100 µl volume. Add 5 ml dye reagent and incubate 5 min. 5. Measure the absorbance at 595 nm. Review Questions 1. What amino acids does the Bradford Protein Assay primarily measure? 2. What color change occurs when proteins combine with Coomassie dye under acidic conditions? 3. What is the standard curve equation for this BSA standard? 4. What is the application of Bradford assay?
Proteins
We generally tend to think of proteins only from a dietary lens, as a component of what we eat. However, they are among the most important and abundant organic macromolecules in the human body, with diverse structures and functions. Every cell contains thousands and thousands of proteins, each with specific functions. Some help in the formation of cellular membrane or walls, some help the cell to move, others act as messages or signals and flow seamlessly from one cell to another, carrying information.
Protein Expression
The method by which living organisms synthesize proteins and further modify and regulate them is called protein expression. Protein expression plays a significant role in several types of research and is highly utilized in molecular biology, biochemistry, and protein research laboratories.
Experiment: Bradford protein assay
give the answers (4-5 lines) of review questions in the end. answer should be logical and understandable and without plagiarism. avoid copy pasting.
Background information:
The Bradford assay is very fast and uses about the same amount of protein as the Lowry assay. It is fairly accurate and samples that are out of range can be retested within minutes. The Bradford is recommended for general use, especially for determining protein content of cell fractions and assessing protein concentrations for gel electrophoresis. The method described below is for a 100 µl sample volume using 5 ml color reagent. It is sensitive to about 5 to 200 micrograms protein, depending on the dye quality. In assays using 5 ml color reagent prepared in lab, the sensitive range is closer to 5 to 100 µg protein. Scale down the volume for the "micro assay procedure," which uses 1 ml cuvettes. Protocols, including use of microliter plates are described in the flyer that comes with the Bio-Rad kit.
Principle:
The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. The assay is useful since the extinction coefficient of a dye-albumin complex solution is constant over a 10-fold concentration range.
Equipment:
In addition to standard liquid handling supplies a visible light spectrophotometer is needed, with maximum transmission in the region of 595 nm, on the border of the visible spectrum (no special lamp or filter usually needed). Glass or polystyrene (cheap) cuvettes may be used, however the color reagent stains both. Disposable cuvettes are recommended.
Procedure:
Reagents
1. Bradford reagent: Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol, add 100 ml 85% (w/v) phosphoric acid. Dilute to 1 liter when the dye has completely dissolved, and filter through Whatman #1 paper just before use.
2. (Optional) 1 M NaOH (to be used if samples are not readily soluble in the color reagent).
The Bradford reagent should be a light brown in color. Filtration may have to be repeated to rid the reagent of blue components. The Bio-Rad concentrate is expensive, but the lots of dye used have apparently been
screened for maximum effectiveness. "Homemade" reagent works quite well but is usually not as sensitive as the Bio-Rad product.
Assay:
1. Warm up the spectrophotometer before use.
2. Dilute unknowns if necessary to obtain between 5 and 100 µg protein in at least one assay tube containing 100 µl sample
3. If desired, add an equal volume of 1 M NaOH to each sample and vortex (see Comments below). Add NaOH to standards as well if this option is used.
4. Prepare standards containing a range of 5 to 100 micrograms protein (albumin or gamma globulin are recommended) in 100 µl volume. Add 5 ml dye reagent and incubate 5 min.
5. Measure the absorbance at 595 nm.
Review Questions
1. What amino acids does the Bradford Protein Assay primarily measure?
2. What color change occurs when proteins combine with Coomassie dye under acidic conditions?
3. What is the standard curve equation for this BSA standard?
4. What is the application of Bradford assay?
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