BISC220/S14: Mod 1 Lab 3

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(Modified Bradford Dye Assay (Bio-Rad™ Assay) for Total Protein)
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[[Image:Series 1 gel loading.jpg]]<br>
{| border="1"
! Lane # !! Sample Name
! 1
| <center>Molecular Weight Standards</center>
! 2
| <center>Commercial β-galactosidase</center>
! 3
| <center>SKIP</center>
! 4
| <center>-IPTG cell extract</center>
! 5
| <center>+IPTG cell extract</center>
! 6
| <center>Crude Extract (CE)</center>
| <center>Purified Fraction (PF)</center>

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Wellesley College     BISC 220     Cellular Physiology

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Purifying and Handling Proteins

The first step in the purification of a specific intracellular protein is extraction from the cells. Bacterial cells can be broken and their enzymes extracted or solubilized by a variety of techniques that may involve mechanical methods (grinding, etc.) or chemical lysis of the cells. The objective is to release the desired enzyme from the cells as gently as possible to retain activity of the enzyme.

Isolation and Purification: There are several methodologies that can be employed for the purification of enzymes, many of which are discussed in your text. These include:

  1. differential solubility
  2. ion exchange chromatography
  3. affinity chromatography
  4. molecular sieve techniques
  5. density gradients
  6. electrophoresis
  7. electrofocusing

Most of these methods rely on differences in either the net charge or molecular weights of the proteins. Generally cruder, less time consuming methods are used in the initial processing of cellular extracts. This is necessary not only because of the large quantities of protein to be processed but also because of the complexity of the protein mixture.


  1. pH: Enzymes have multiple charges on their surfaces, which must be preserved to maintain the native 3-D structure and hence enzymatic activity. Buffers are used to maintain the enzyme solutions at a desired pH. The buffer must be in the appropriate concentration, have the correct pKa, and must not adversely affect the protein. See your general chemistry text for a review of buffers and other inorganic chemistry terms.
  3. Protection Against Sulfhydryl Oxidation: Enzymes may contain many sulfhydryl groups (SH). One or more may be required for the activity of an enzyme. If these sulfhydryl groups become oxidized, they form intra- or intermolecular disulfide bonds. If necessary, the most effective method of retarding such oxidation is the addition of a reducing agent to the buffer. Dithiothreitol (DTT) and β-mercaptoethanol (βME) are among the most common and effective reducing agents. β-galactosidase seems to be most stable in a reducing environment, so often β-mercaptoethanol is included in the solutions used to extract and assay the enzyme.
  4. Protection Against Heavy Metals: In addition to oxidation, sulfhydryl groups may react with heavy metal ions such as lead, iron or copper. Principal sources of metal ions are the reagents used to make up buffers, substrates, and water itself. Deionized or distilled water is used to make up reagents and a chelating agent such as EDTA (ethylenediaminetetraacetic acid) can also be added.

Basic Rules for Handling Enzymes

The following information is taken from a popular primer now published by Roche Pharmaceuticals, but first published in the December 1985 issue of BMBiochemica. by Boehringer Mannheim Biochemicals.

  3. Enzymes should be handled in the cold (0-4°C) at all times. Dilute for use with ice-cold buffer or distilled water. While using an enzyme solution or suspension at the bench, keep it in on ice.
  5. Enzymes, especially those that have been diluted, should be periodically checked for activity to ensure that any loss in activity is taken into account when designing an experimental protocol. Expiration dates on vials only refer to enzymes stored in the original form at the correct temperature.
  8. Vials containing lyophilized enzymes (as well as cofactors such as NADH and NADPH) should be warmed to room temperature before opening. This prevents condensation of moisture onto the powder, which can cause loss of activity or degradation. If the reagent is hygroscopic, mishandling may ruin the entire vial.
  9. Avoid repeated freeze-thawing of dilute enzymes and lyophilizates in solution. Store in small aliquots. Thaw one portion at a time and store that portion once thawed at 4°C. The stability of individual enzymes may vary greatly and often should be determined empirically under your exact conditions.
  10. Detergents and preservatives should be used with caution, since they may affect enzyme activity. Sodium azide, for example, inhibits many enzymes which contain heme groups (e.g. peroxidase). Detergents added at concentrations above their critical concentration form micelles which may entrap and/or denature the enzyme.
  11. Enzymes should be handled carefully. To avoid contamination of any kind, use a fresh pipette tip for each aliquot that is removed from the parent vial. Never return unused material to the parent vial. Wear gloves to prevent contaminating the enzyme with proteases, DNAses, RNAses, and inhibitors often found on fingertips. Never pipette by mouth.
  12. Adjust the pH of the enzyme buffer at the temperature at which it will be used. Many common buffers (Tris, glycylglycine, Bes, Aces, Tes, Bicine, Hepes) change rapidly as the temperature changes. For instance, Tris buffer decreases 0.3 units of pH for EVERY 10°C rise in temperature. A solution of Tris, adjusted to pH7.5 at +25°C will have a pH of 8.1 at 4°C or 7.2 at 37°C. The change in pH per 10°C temperature change for other buffers is: Aces, -0.20; Bes, -0.16 ; Bicine,-0.18; glyclyglycine, -0.28; Hepes,-0.14; Tes, -0.20 [Good, N.E., Winget, G.D., Winter, W., Connolly, T.N., Izawa, S., and Singh, R.M. (1966). Hydrogen ion buffers for biological research. Biochemistry 5, 467-77.]
  13. The absorbance at 280nm is widely used to quickly determine the protein concentration of an enzyme solution. However, this absorbance is due to the presence of tyrosine and tryptophan in the protein. If an enzyme (e.g. superoxide dismutase) contains low amounts of these two amino acids it will not absorb significantly at 280nm.

Resources: Detailed information is available on many enzymes. The following are excellent resources.

Affinity Chromatography

The use of metal chelate affinity chromatography for protein purification was first reported by Porath and colleagues in 1975. This landmark report applied the knowledge that histidine and cysteine form rather stable complexes with some cations such as zinc and copper ions. We now know that the amino acid tryptophan shares this characteristic with histidine and cysteine and that some proteins have specific binding sites for these metals. Porath et al. (1975) devised a method to tightly bind metal ions to a solid matrix such as agarose beads. The matrix resides in a column through which protein solutions are passed. Some proteins in these solutions bind to the matrix but can be specifically eluted by a low pH wash solution. Later work (Porath and Olin, 1983; Kagedal, 1998) showed that it is possible to release proteins from such columns using a strong soluble chelator such as EDTA or by chemically competing with the binding. For example, histidine binding can be disrupted by including imidazole in the protein elution buffer. The chemical structure of imidazole is similar to the ring structure of histidine, therefore it competes for the binding site on the cation matrix.

In today’s lab, we are using agarose beads chelated with nickel to fractionate the 6xHis-tagged β-galactosidase. Nickel effectively binds histidine, and the combination of 6 histidine residues in a row at the amino terminus of β-galactosidase should lead to rather tight binding. After washing the nickel chelated agarose to remove any unbound or loosely bound protein, we will use a buffer containing 200 mM imidazole to release the 6xHis-tagged enzyme from the nickel-chelated agarose beads.

Affinity Column Purification Protocol

In this laboratory session you will purify β-galactosidase from the cell pellet of the genetically modified E. coli you induced last week to overexpress this enzyme.


Microsoft Word File: Purification of 6xHis-tagged β-gal

  1. Defrost the frozen pellet of E.coli BL21(pET-14b) cells prepared in the previous lab session and add 10.0 mL of B-Per Protein Extraction Reagent™, a mild anionic detergent (the exact ingredients of which are proprietary), to the centrifuge bottle. Add 20μL of DNAase (to achieve a concentration of 1 unit/mL from the 5000units/ml stock) to the B-Per and cells. Resuspend the cells by vortex mixing and pipetting up and down until the pellet is off the side of the bottle and all clumps are gone. Try to avoid bubbles. This should take about 5 minutes total, of alternately mixing and letting the mixture sit in your ice bucket. Your goal is to dissolve the cell walls and membranes which will lyse the cells and allow degradation of the DNA. Ask your instructor to check your suspension before proceeding to step 2. If you have obvious clumps, the cells are less likely to be lysed sufficiently.
  3. Carefully pour the entire suspension into a Corex tube fitted with a rubber adapter and centrifuge for 15 minutes at 10,500 rpm in the Sorval refrigerated centrifuge at 4°C using an SS-34 rotor. You will need to make a balance tube with water. Be sure and record in your lab notebook the speed as g force (use the chart near the centrifuges to make the conversion).
  4. Pour the supernatant only into a 15 mL graduated conical tube. This solution of dissolved proteins in B-Per reagent is your cell-free or Crude Extract (CE). The pellet is bacterial cell debris that can be dumped into your waste container. The Corex tubes are NOT disposable. Please rinse them out and leave them on your bench or return them to your instructor.
  5. Determine and record the total volume of the crude extract (CE) using the volume markings on the tube. Pipet 600 µL of CE into a microcentrifuge tube labeled with your team color, lab day, initials and "CE" or “Crude Extract.” Store the aliquot on ice. At the end of lab today, you will give what remains of this aliquot to your instructor to freeze for use next time.
  6. Add 1000µL (1mL) of the Nickel-Chelated Agarose to the volume of CE remaining in the 15mL conical tube. It is essential that the agarose suspension be properly mixed before removing an aliquot, so mix it well by swirling or gentle vortexing just before removing it from the stock.
  8. Using a Pasteur pipette, carefully remove and discard the supernatant solution into the provided waste container. Do not try to pour off the supernatant! The 6xHis-tagged proteins should now be bound to the nickel-chelated agarose beads at the bottom of the tube.
  9. Add 3.0 mL of Wash Buffer #1® to the agarose beads in the conical tube, and resuspend using a pipette. Do not vortex. Gently shake the suspension at room temperature for 5 minutes on the platform shaker. Centrifuge for 3 minutes in a clinical, benchtop centrifuge. Carefully remove supernatant and discard.
  12. Place the spin column in a new collection tube and add 500µL of Elution Buffer containing imidazole. Mix the agarose and the buffer in the spin column using the NOT DISPOSABLE plastic mixer provided by your instructor. Incubate the spin column at room temperature for 5 minutes. During this time, most of the 6x His-tagged protein will be eluted from the nickel-chelated agarose into the buffer. After the 5 minute incubation period, recover the protein by centrifuging for 2 minutes at top speed in a microcentrifuge. Your purified fraction will be the flow-through in the collection tube. If you have considerably less than 500µL, respin the column to collect more volume.
  13. Take the spin column out of the collection tube and discard the spin column. Do not discard the material in the collection tube. Using a Pasteur pipette, transfer all of the contents containing the purified β-galactosidase to a new microfuge tube with volume markings. Record the volume in your notebook! Label the tube to indicate that this is the purified β-galactosidase fraction (e.g., "PF β-gal"), and save it in your ice bucket.


You are now ready to assay both the Crude Extract and Purified Fraction for total protein, using a modified Bradford Dye Assay (Bio-Rad assay). You will later assay both CE and PE for β-galactosidase activity (enzyme function) using a different assay.


We will now determine the total protein concentration in our CE and PF fractions in order to load each lane of our gel with equal amounts of protein. Loading lanes equally is the ONLY way we can make comparisons between lanes.

Microsoft Word File: Media:Modified Bradford Dye Assay Protocol.doc

Testing the quality of the purification:

Dilution Preparation for the Standard Curve


Tube # BSA Concentration Z Buffer in μL Stock BSA
0.1 mg/ml
0.2 mg/ml
0.4 mg/ml
0.6 mg/ml
0.8 mg/ml
1.0 mg/ml


Dilution Preparation of Unknowns:


      Amount of Z-buffer required = ?

      Amount of CE or PF required = ?

Total Protein Assay Protocol:

Tube # BSA Standard Diluted in Z-Buffer CE (1:5 dilution in Z-Buffer) PF (1:5 dilution in Z-Buffer) Z-Buffer Protein Assay Reagent™
1 0.1 mg/ml (100 ul) - - - 5 ml
2 0.2 mg/ml (100 ul) - - - 5 ml
3 0.4 mg/ml - - - 5 ml
4 0.6 mg/ml - - - 5 ml
5 0.8 mg/ml - - - 5 ml
6 1.0 mg/ml - - - 5 ml
7 - 100 ul - - 5 ml
8 - 100 ul - - 5 ml
9 - - 100 ul - 5 ml
10 - - 100 ul - 5 ml
11 - - - 100 ul 5 ml



Polyacrylamide Gel Electrophoresis


  3. 2% SDS Sodium dodecyl sulfate,
  5. in the solvent 0.0625 M Tris buffer pH 6.7



Microsoft Word File: Media:Gel Electrophoresis Protocol GelCode.doc



Lane # Sample Name
Molecular Weight Standards
Commercial β-galactosidase
-IPTG cell extract
+IPTG cell extract
Crude Extract (CE)
Purified Fraction (PF)

Preserving the Enzymatic Activity of the CE and PF

Glycerol acts to preserve enzymatic activity while the samples are frozen. We will be using these aliquots of crude extract and purified fraction to study the enzyme kinetics of β-galactosidase in Lab 4.

  1. Determine the remaining volume of the purified fraction and add an amount of 70% glycerol equal to 1/2 the volume of that fraction. Mix well by inversion. For example, if your purified fraction has a volume of 250 µl, you would add 125 µl of 70% glycerol solution it.

  2. Pipette 750 ul of Crude Extract (CE) into a clean microfuge tube. Label it CE. Add 375 μL of 70% glycerol to this fraction. Mix well by inversion.
  3. Give both labeled tubes to your instructor to save for next week.


  2. Using the standard curve generated in the previous step, calculate the molecular weight of the band(s) that you have identified as β-galactosidase. What is the calculated molecular weight?



Background information on enzymes and lab techniques:


Kågedal, L. (1998) Immobilized Metal Ion Affinity Chromatography. In: Protein Purification (Janson, J. C. and Rydén, L., eds.), Wiley-VCH, New York, NY, pp. 311–342.





Specific References for β-galactosidase:




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