Ateneo de Zamboanga University College of Nursing NURSING SKILLS OUTPUT (NSO) Protein Assay Report No.

Description: It is a spectroscopic analytical procedure used to measure the concentration of protein in a solution. It is subjective, i.e. dependent on the amino acid composition of the measured protein. Materials/Equipment Needed:

Lyophilized bovine plasma gamma globulin or bovine serum albumin (BSA) Coomassie Brilliant Blue 1 0.15 M NaCl Spectrophotometer and tubes Micropipettes

Procedures: Procedure (Standard Assay, 20-150 g protein; 200-1500 g/ml) 1. Prepare a series of protein standards using BSA diluted with 0.15 M NaCl to final concentrations of 0 (blank = NaCl only), 250, 500, 750 and 1500 g BSA/mL. Also prepare serial dilutions of the unknown sample to be measured. 2. Add 100 L of each of the above to a separate test tube (or spectrophotometer tube if using a Spec 20). 3. Add 5.0 mL of Coomassie Blue to each tube and mix by vortex, or inversion. 4. Adjust the spectrophotometer to a wavelength of 595 nm, and blank using the tube which contains 0 BSA. 5. Wait 5 minutes and read each of the standards and each of the samples at 595 nm wavelength. 6. Plot the absorbance of the standards vs. their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples. Procedure (Micro Assay, 1-10 g protein) 1. Prepare standard concentrations of BSA of 1, 5, 7.5 and 10 g/mL. Prepare a blank of NaCl only. Prepare a series of sample dilutions. 2. Add 100 L of each of the above to separate tubes (use microcentrifuge tubes) and add 1.0 mL of Coomassie Blue to each tube. 3. Turn on and adjust a spectrophotometer to a wavelength of 595 nm, and blank the spectrophotometer using 1.5 mL cuvettes. 4. Wait 2 minutes and read the absorbance of each standard and sample at 595 nm. 5. Plot the absorbance of the standards vs. their concentration. Compute the extinction coefficient and calculate the concentrations of the unknown samples.

October 7, 2010 Date

Mr. Julius C. Lapasaran, RN Clinical Instructors Initials Kassandra Kristel G. Andrade Page 1 of 2

Diagram/Illustration

NURSING RESPONSIBILITIES

Before Procedure

Explain to the patient the procedure. Make sure that the signs the consent form before the test.

During Procedure
Maintain aseptic technique.

After Procedure
Provide patient comfort. Tell patient that results will be subjected for study and obtained soon.

References: http://en.wikipedia.org/wiki/Bradford_protein_assay http://books.google.com/books?id=4UUKvwMirD4C&pg=PA120&dq=bradford+protein+assay&hl=en&ei =aSysTNOqD46cbaJ4bUE&sa=X&oi=book_result&ct=result&resnum=2&ved=0CDgQ6AEwAQ#v=onepage&q=b radford%20protein%20assay&f=false http://www.gbiosciences.com/SPNproteinassays-desc.aspx

October 7, 2010 Date

Mr. Julius C. Lapasaran, RN Clinical Instructors Initials Kassandra Kristel G. Andrade Page 2 of 2