Quantitation of Total Protein in Extracts

The following is a method to quantify total protein in extracts using Coomassie Plus Protein Assay Reagent (Pierce).

1. Pipet 998 ^L of H2O into appropriately labeled tubes that can hold more than 2 mL liquid.

2. Add 2 ^L of protein extract to each tube. Make a blank control by adding 2 ^L of EB instead. To build a standard curve, prepare 3 to 5 samples by adding 2 ^L of a BSA solution at known concentration (e.g., 0.05, 0.2, 0.5, and 2 mg/mL) to the tubes.

3. Add 1 mL Coomassie Plus solution to each tube and mix well.

4. Set a spectrophotometer at 595 nm and calibrate the "zero" using the blank.

5. Measure the absorbance of the samples. If BSA controls were used, create a standard curve to determine the protein concentration of the samples (see Note 5).

4. Notes

1. EB is made using the following stock solutions:

a. 500 mM HEPES: dissolve 23.8 g of HEPES (free acid form) in 200 mL of H2O, adjust pH to 7.5 with HCl or NaOH, filter, and store at 4°C.

b. 4 M NaCl: add 58.4 g of NaCl to 250 mL of H2O and filter.

c. 10% Triton X-100: dissolve 10 mL of Triton X-100 in 90 mL H2O.

d. 1 M DTT: dissolve 1.54g of DTT in 10 mL of 20 mM sodium acetate, pH 5.2, filter, make 1 mL aliquots, and store at -20°C.

e. 1 M sodium p-glycerophosphate in H2O.

f. 0.5 M sodium orthovanadate: make 500 mM solution in H2O, adjust pH to 10.0 with HCl or NaOH, boil the solution until it turns colorless, and let it cool down to room temperature. Measure pH. If pH has changed significantly, readjust pH to 10.0 and boil again until it becomes colorless. Repeat this step until pH stabilizes near 10.0. Make 1-mL aliquots and store them at -20°C.

g. 500 mM EDTA: make 500 mM solution in H2O and filter.

h. 100 mM PMSF: make 100 mM solution in isopropanol and store at -20°C.

i. 10 mg/mL aprotinin: dissolve 10 mg in 1 mL H2O and store at -20°C. j. 5 mg/mL leupeptin: dissolve 5 mg in 1 mL H2O and store at -20°C.

k. 2 mg/mL pepstatin: dissolve 2 mg in 1 mL ethanol and store at -20°C.

2. DTT (reducing agent), sodium p-glycerophosphate (general phosphatase inhibitor), sodium orthovanadate (tyrosine phosphatase inhibitor), EDTA (chelant) and PMSF, aprotinin, leupeptin, and pepstatin (protease inhibitors) need to be added to the EB immediately prior to use.

3. When transferring the pulverized material into a microcentrifuge tube, make sure not to add more than the equivalent of 100 ^L of tissue, as the sample may splash out during homogenization. Select a round-bottom tube so that the pestle (used in step 3) can touch the bottom of the tube. This facilitates complete homogeniza-tion of the tissue.

Fig. 1. Determination of the efficiency of protein extraction. Proteins were extracted from mouse liver collected at two time points (9 and 21 h after lights-on in a 12-h light:12-h dark environmental cycle). Tissue debris (pellet) after the first centrifuga-tion were resuspended in 1X sample buffer in the same volume as the supernatant (sup), boiled for 3 min at 95°C, and sonicated briefly. Both the supernatant and the resuspended pellet were run on the same gel, blotted, and immunoassayed with anti-mPER1 antibodies to assess the efficiency of the extraction condition. The arrows indicate nonspecific bands. mPER1 and the top nonspecific protein were extracted efficiently, whereas only a minor portion of the bottom nonspecific protein was extracted under these conditions.

Fig. 1. Determination of the efficiency of protein extraction. Proteins were extracted from mouse liver collected at two time points (9 and 21 h after lights-on in a 12-h light:12-h dark environmental cycle). Tissue debris (pellet) after the first centrifuga-tion were resuspended in 1X sample buffer in the same volume as the supernatant (sup), boiled for 3 min at 95°C, and sonicated briefly. Both the supernatant and the resuspended pellet were run on the same gel, blotted, and immunoassayed with anti-mPER1 antibodies to assess the efficiency of the extraction condition. The arrows indicate nonspecific bands. mPER1 and the top nonspecific protein were extracted efficiently, whereas only a minor portion of the bottom nonspecific protein was extracted under these conditions.

4. When extracting a protein for the first time, it is important to assess the efficiency of extraction, because potential pour solubility in the EB used can seriously bias any downstream application. To determine the efficiency of extraction a comparison is made (by Western blot) between the soluble (in the supernatant) and the insoluble (in the pellet) fractions (Fig. 1). First the pellet is washed with EB to remove any remaining soluble protein, then the insoluble fraction is extracted from the tissue debris with 1X sodium dodecyl sulfate (SDS) sample buffer (diluted from a 2X SDS sample buffer stock: 100 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 5% 2-mercaptoethanol, 2 mM EDTA, 0.1 mg/mL bro-mophenol blue). To remove the soluble proteins, resuspend the pellet in 5 to 10 vol of EB, spin at 12,000g at 4°C for 5 min, and remove the supernatant. To extract the insoluble fraction add 3 to 5 vol of 1X SDS sample buffer (the high concentration of SDS and 2-mercaptoethanol does solubilize most proteins except cytoskeletal proteins) to the tissue debris and homogenize as described in the protein extraction methods. Sonicate the sample if it is too viscous. Before loading on the gel, heat the sample at 95°C for 3 min, and centrifuge at 12,000g for 5 min. If a minor fraction of the protein was extracted, increase the concentration of detergent and salt in the EB and/or change the homogenization method. 1X SDS sample buffer can be directly used to extract proteins for Western analysis. In this case, however, it is difficult to measure protein concentration.

5. If the same protein quantitation method is used repeatedly, it is not necessary to include the BSA controls for every experiment. The BSA controls need to be done for only the first two or three experiments. Absorbance data of samples can be used to estimate amounts of total protein present in the samples based on previous standard curves.

References

1. Lee, C., Etchegaray, J. P., Cagampang, F. R. A., Loudon, A. S. I., and Reppert, R. M. (2001) Posttranslational mechanisms regulate the mammalian circadian clock. Cell 107, 855-867.

2. Braford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

Was this article helpful?

0 0

Post a comment