344
Related Techniques
Results
Cell number can be estimated from the predetermined relationship between cell number and absorbance. It is prerequisite to determine relationship between cell number counted with hemocytometer and absorbance for each cell line.
Subprotocol 4
Protein Measurement
Protein content is often used for estimating total cellular material, and
can be used in growth experiments. There are various methods available
for measuring protein content. Of these methods, the method of Bradford
(1976) has been used widely, and the reagents are available as a kit from
Bio-Rad.
Materials
Equipment - Spectrophotometer
Solutions - Coomassie brilliant blue solution
Protein
staining
Spectrophotometry
- Dissolve 100 mg Coomassie brilliant blue G-250 in a mixture of
50 ml of 95% ethanol and 100 ml of 85% phosphoric acid
- Dilute this mixture to 1,000 ml using distilled water
Procedure
1. Solubilize cells (approximately 10 6 ) in 0.3 N NaOH and 1% sodium
lauryl sulfate for 30 min at room temperature.
2. Add 1.0 ml Coomassie brilliant blue solution to 100 fll protein solution, mix, and leave for 10 min.
1. Read the absorbance on spectrophometer at 595 nm against a reagent
blank and compare with bovine serum albumin (BSA) standard curve
(1-5 flglml BSA).
2. Referring to the standard curve, determine the protein content in the
sample.
Results
Cell number can be estimated from the predetermined relationship between cell number and protein content. The relationship between cell
number and protein content should be determined for each cell line.
Related Techniques
Results
Cell number can be estimated from the predetermined relationship between cell number and absorbance. It is prerequisite to determine relationship between cell number counted with hemocytometer and absorbance for each cell line.
Subprotocol 4
Protein Measurement
Protein content is often used for estimating total cellular material, and
can be used in growth experiments. There are various methods available
for measuring protein content. Of these methods, the method of Bradford
(1976) has been used widely, and the reagents are available as a kit from
Bio-Rad.
Materials
Equipment - Spectrophotometer
Solutions - Coomassie brilliant blue solution
Protein
staining
Spectrophotometry
- Dissolve 100 mg Coomassie brilliant blue G-250 in a mixture of
50 ml of 95% ethanol and 100 ml of 85% phosphoric acid
- Dilute this mixture to 1,000 ml using distilled water
Procedure
1. Solubilize cells (approximately 10 6 ) in 0.3 N NaOH and 1% sodium
lauryl sulfate for 30 min at room temperature.
2. Add 1.0 ml Coomassie brilliant blue solution to 100 fll protein solution, mix, and leave for 10 min.
1. Read the absorbance on spectrophometer at 595 nm against a reagent
blank and compare with bovine serum albumin (BSA) standard curve
(1-5 flglml BSA).
2. Referring to the standard curve, determine the protein content in the
sample.
Results
Cell number can be estimated from the predetermined relationship between cell number and protein content. The relationship between cell
number and protein content should be determined for each cell line.