adding microbiuret

SAMPLE LAYOUT OF AN EXPERIMENT:
PROTEIN CONCENTRATION IN UNKNOWNS BY MICROBIURET

©David B. Fankhauser, Ph.D.,
Professor of Biology and Chemistry
University of Cincinnati Clermont College,
Batavia OH 45103
 tubes ready to read
Adding microbiuret 

This page has been accessed Counter times since 14 October 2002. 
4 Nov 1992, rvsd 12 Oct 94, 17 Sept 95, 23 Sept 96, 15 Oct 96, 12 Oct 99, 11 Oct 00, 9 Oct 01.

From DBF's Hopkins Notebooks, III, p. 102 & VI, p. 75.
tubes ready to read

See Protein Assay by Microbiuret:  Standardization for introduction, and how to standardize the microbiouret solution.
 
1. PLAN YOUR EXPERIMENT, WRITE OUT YOUR EXPERIMENT TABLE:
Calculate the dilutions of unknowns needed to bring their protein concentrations down to approximately 1-5 mg/mL. Plan two tubes per each diluted sample, one with 0.1 mL, the other with 1.0 mL. Calculate and record the amount of water required for each tube to  q.s. to 2.0 mL.  Create a table similar to one in the standardization with these nine columns:

EXAMPLE EXPERIMENTAL TABLE:

tube#
specimen being tested
 dilution
factor
 mL
H 2O
 sample
aliquot
 mL
microbiuret
 A 325
mg protein
per tube
 mg protein
in 100 g sample
Blank
 distilled water
 n/a
 2.0
 n/a
 2.0
                  
                      

1
 saliva, student 1
  5x
 1.9
  0.1
 1.0
          
  
  
2
 saliva, student 1
  5x
 1.0
 1.0
 1.0
  
 
  
3
 saliva, student 2
 5x
 1.9
 0.1
 1.0


4
 saliva, student 2
 5x
 1.0
 1.0
 1.0


5
 solid (i.e., dog food or cat food)
 100x
 1.9
 0.1
 1.0
  
 
  
6
 solid (i.e., dog food or cat food)
 100x
 1.0
 1.0
 1.0


7
 liquid 1 (diluted egg yolk or white, milk, etc)
 50x
 1.9
 0.1
 1.0


8
 liquid 1 (diluted egg yolk or white, milk, etc)
 50x
 1.0
 1.0
 1.0


9
 liquid 2 (diluted egg yolk or white, milk, etc)
 50x
 1.0
 0.1
 1.0


10
 liquid 2 (diluted egg yolk or white, milk, etc)
 50x
 1.9
 1.0
 1.0


11
 standard mg/mL protein (BSA)
 n/a
 1.6
 0.4
 1.0
  
 
  
12
 standard mg/mL protein
 n/a
 1.2
 0.8
 1.0
  
 
  

Include a blank, as in the standardization procedure, and  standardization tubes with 0.5 and 1.0 mg standard protein each. 

2. PREPARE SAMPLE: DILUTE, SUSPEND OR DISSOLVE:

The final concentration of protein in the diluted samples should be between 1 to 5 mg/mL. 

Solids: For a 1% suspension: weigh out 300-500 mg. Grind very fine in mortar and pestle. Add a few drops dH 2O, grind to paste, add few more drops, make slurry, wash grindings into graduated cylinder, q.s. to 100x weight with dH 2O (i.e., 30 mL for 300 mg solid).

Liquids: For concentrated fluids ( egg white or yolk, blood, milk, etc) make a 1:50 dilution : add 0.1 mL to 4.9 dH 2O. Collect saliva in 10 mL beaker, dilute 1:5 (0.4 mL + 1.6 mL dH 2O .  For dilute biological fluids like urine, use undiluted as a first approximation. Dilute protein-rich materials 200x , saliva 5x. Vortex thoroughly after the diluting. 

3. SET UP TUBES, ADD dH 2O TO TUBES AS IN YOUR TABLE: 

Set up the appropriate number of labeled, clean 13 x 100 mm test tubes in a rack (2 tubes/sample). Add the dH2 O first [then the protein sample,  finally the microbiuret reagent].

4. ADD PROTEIN ALIQUOTS TO THE SET OF TUBES:

Carefully following your protocol table, add the prescribed amounts of standard protein to the standardization tubes, and 0.1 and 1.0 mL aliquots of diluted specimens to their tubes. Samples should always be added just below the surface of the water.

5. ADD 1 mL OF MICROBIURET TO EACH TUBE.

Make a visual check to see that all tubes appear to have a identical final volume of 2 mL in them (water + sample).  Then add 1.0 mL of microbiuret reagent by repipet, mix well by vortex, let sit for 15 min.

6. READ ABSORBENCY AT 325 nm:

Use the B tube (containing no protein) as the blank, determine the A 325 of each tube in succession. You may not need to wash the cuvette between samples, but drain it thoroughly, touching off the last drop from the cuvette on a paper towel prior to adding the next specimen to minimize cross contamination. 

7. CALCULATE THE CONCENTRATION OF PROTEIN:

Calculate how much protein is in each tube using the conversion factor from the previous lab (A 325 of the specimen x conversion factor). Do the two standard tubes agree with the standardization? Calculate the concentration in the original sample: 

 

mg/mL in original sample = A 325 x conversion factor x 1/aliquot used x dilution factor

 

 

HINTS FOR SET UP:

Prepare a table of data of the results from the entire class.
 

Set up tables with prepared suspensions/dilutions of dog food, cat food, milk, etc.
Place pipeters with 10. And 0.1 volumes at each table, rotate around with tubes having appropriate volume of water in them.

Used Eppendorf Repipeter for distribution of MB.