How do you do a BSA standard curve?
To create a standard curve using your BSA standards, pipette 20 μL of each standard into an eppendorf tube. Add 980 µL of the prepared dye reagent to each tube and vortex briefly. Incubate the tubes at room temperature for 5 to 50 minutes.
What is the standard curve equation?
The equation y=mx+b can be translated here as “absorbance equals slope times concentration plus the y-intercept absorbance value.” The slope and the y-intercept are provided to you when the computer fits a line to your standard curve data. The absorbance (or y) is what you measure from your unknown.
What is BSA standard curve?
A standard curve was developed using a series of Bovine Serum Albumin (BSA) standards in the 100 μg/ml to 1,500 μg/ml range. The absorbance of each sample was measured at 595 nm and plotted versus [BSA]. The resulting line was fit by the linear least squared method.
How do you calculate BSA concentration?
The concentration of BSA in solution can be determined by substituting the molecular weight, extinction coefficient and λmax into a derived form of the Beer-Lambert Law. A substance’s λmax is the wavelength at which it experiences the strongest absorbance. For BSA, this wavelength is 280 nm.
How will you prepare a 5% BSA solution?
For example, to make a 100mL 3% BSA solution, use 3 grams of BSA and bring the volume to 100mL. To make a 5% solution of BSA in 500mL of media (which is the typical media bottle size) 25grams of BSA would be used, brought to a volume of 500mL with the media of interest.
What is the purpose of a BSA standard?
In molecular biology, BSA is used to stabilize some restriction enzymes during digestion of DNA and to prevent adhesion of the enzyme to reaction tubes, pipet tips, and other vessels.
What is a good standard curve?
In general, a good standard curve should have the following characteristics: R-squared value is greater than 0.95, and as close to 1 as possible. The OD of the blank well should be lower than 0.25. The maximum absorbance value should be higher than 0.8.
Why is BSA a good standard?
BSA is used because of its stability to increase signal in assays, its lack of effect in many biochemical reactions, and its low cost, since large quantities of it can be readily purified from bovine blood, a byproduct of the cattle industry.
Why is BSA used for standard curves?
The bovine protein standard is the preferred standard in protein assays because in addition to its ability to increase signal in assays, bovine serum is affordable and easily mass-producible.
Can I autoclave BSA solution?
It will stick if you use pure BSA. If you dilute it, it is possible. i would heat 10′ 37° the BSA solution and then flter it.
Why does the BSA curve vary between assays?
Therefore, it depends on the reactant concentration, temperature, and pH of the sample, as well as any other condition that could affect the reaction, including the specific buffer and salts included in the reagent. If the reagent concentration is different between assays, the results could be different.
How to calculate sample concentration on the standard curve?
To calculate the sample concentration based on the standard curve, first you find the concentration for each sample absorbance on the standard curve; then you multiply the concentration by the dilution factor for each sample.
What are the requirements for a standard curve?
It is necessary to generate a standard curve for both the target gene and the reference gene each time the assay is run. Each of the standard curves must meet certain criteria, including R > 0.98, a slope of −3 to −4, and a defined C t for a neat sample.
Is it necessary to dilute a standard curve?
It may be necessary to dilute test samples to ensure that their mean fluorescence values fall within the limits or range of the generated standard curve. For best results, samples that are known or assumed to contain high levels of a given protein should be diluted as described below. 1.