Laboratory Exercise 4: Techniques for Protein Isolation

Question 1

In exercise 4, the crude ovalbumin will be isolated from egg white __ and the __ of casein will be determined by __.

Answer 1

• ammonium sulfate precipitation
• isoelectric pH
• isoelectric precipitation

Question 2

Materials in laboratory exercise 4: techniques for protein isolation

Answer 2

• Beaker, 250 mL (2)
• Beaker, 50 mL (2)
• Graduated cylinder, 50 mL (2)
• Pipet, 5 mL
• Stirring rod
• Spatula
• Filter funnel (2)
• Filter paper (5)
• Wash bottle

Question 3

Chemicals in exercise 4: techniques for protein isolation (Part 1)

Answer 3

• 1.7 M Acetic acid
• Ammonium sulfate powder
• Ethyl ether
• Ethanol

Question 4

Chemicals in exercise 4: techniques for protein isolation (Part 2)

Answer 4

• 1 M NaOH
• 1.7 M Acetic acid
• 0.1 M glycine-HCl buffer (pH 2.7)
• 0.1 M acetate buffer (pH 4.7)
• 0.1 M phosphate buffer (pH 6.7)

Question 5

• are the most widely studied among the four major biomolecules.
• a lot of isolation and purification protocols have already been tried and proven to be efficient in terms of both the quality and quantity of protein isolates obtained.
• techniques and assays regarding the determination of its molecular weight, number of subunits, concentration in solution, and biological activity have already been established.

Answer 5

proteins

Question 6

• is one of the most common methods by which proteins are isolated from a mixture of soluble substances.
• It can be achieved by taking advantage of some properties of proteins such as solubility.

Answer 6

proteins precipitation

Question 7

Two techniques are commonly used in protein precipitation

Answer 7

• salting-out process
• isoelectric precipitation

Question 8

The salting-out process usually precipitated by saturating the protein with

Answer 8

• ammonium sulfate
• magnesium sulfate

Question 9

is a function of the ionic strength of the solution, where the ionic strength depends not only on the concentrations of the cations and anions but also on the electrical or valence charge of each ion.

Answer 9

The solubility of a protein in a salt solution

Question 10

• Solubility of the protein __ as the ionic strength __ and the protein eventually __.
• This is because when the ionic strength of the protein solution is __ water tends to interact more with the ions from the added salt thereby __ water-protein interactions.

Answer 10

• decreases
• increases
• precipitates completely
• increased
• weakening

Question 11

• When the ionic strength of the protein solution is increased, proteins are left to interact with one another resulting in the formation of __.
• The precipitate may contain __ and biology __. In some cases, the proteins become __.

Answer 11

• protein aggregates
• properly folded
• denatured

Question 12

• In protein aggregates, if proteins become denatured, biological activity may be recovered when the __ is decreased.
• The proteins in a mixture may differ in their solubility at a given __ and may precipitate at different levels of __.

Answer 12

• salt concentration
• salt concentration
• ionic strength

Question 13

the albumins and globulins of egg white can be separated if the egg white solution is only half saturated with respect to the __; the __ will precipitate but the __ will not.

Answer 13

• ammonium sulfate
• globulins
• albumins

Question 14

• Variation in __ changes the state of ionization of the functional groups in the protein and eventually its net charge.
• Generally, protein solubility is __ when it is in a solution with pH equal to its isoelectric (IpH) and __ when the solution’s pH is on either side of the protein’s IpH.

Answer 14

• pH changes
• least
• increases

Question 15

• When the pH of the solution is __ to the protein’s IpH, the net charge of the protein is __ favoring __ thus the protein __ out of the solution
• This is the basis for the isolation technique called __.

Answer 15

• equal
• zero
• protein-protein interactions
• precipitates
• isoelectric precipitation

Question 16

Procedure 1-3 in Exercise 4

Answer 16

1. Isolation of crude ovalbumin from egg white by ammonium sulfate precipitation (salting out).
2. Isolation of Casein
3. Isoelectric precipitation of casein

Question 17

Specifics: Procedure 1 (Exercise 4)

Break up __ eggs and separate the egg whites carefully from the yolk. Stir the egg whites to break up the membrane and filter through __. Measure 40.0 mL of the filtered undiluted egg white. Add 0.06 mL of __ for every 1.0 mL of egg white slowly with constant stirring. Remove the precipitate formed using a cheesecloth.

Obtain 30.0 mL of the filtrate and add 7.26 g __ portion by portion with constant stirring to bring the solution to 40% saturation. Take note of any noticeable change. Filter through cheesecloth. Bring the solution from the 40% to 60% saturation by adding 3.90 g __ in the same manner as the previous addition. Allow the solution to stand with occasional stirring for 30 minutes in an __ and then filter using a pre-weighed filter paper. Air dry and weigh the precipitate. Put the dried precipitate in a labelled vial and store in the refrigerator. Calculate the amount of __ isolated in the refrigerator. Calculate the amount of crude ovalbumin isolated and report the yield as __

Answer 17

• two medium-sized
• cheesecloth
• 1.7 M acetic acid
• powdered ammonium sulfate
• powdered ammonium sulfate
• ice bath
• crude ovalbumin
• % (weight dried precipitate/volume egg white).

Question 18

Specifics: Procedure 2 (Exercise 4)

Weigh out 5 grams of __ and dissolve it in 20 mL of warm water (pre warmed to 38˚C) in a 100 mL beaker. Bring the temperature of the solution to 55˚C (do not exceed) on a hot plate, remove the thermometer, and then add dropwise a solution of __ while stirring with a stirring rod. Do not add the acid too rapidly. Continue the acid addition (slightly less than 1.2 mL will be required), keeping the beaker on the hot plate, until the liquid changes from milky to almost clear and the casein no longer separates. Stir the precipitated casein until it forms a large amorphous mass; then remove it with a stirring rod or tongs and place it in another beaker.

Collect the casein by filtration to remove as much water as possible. Press the solid with a spatula. Place the casein in a 100 mL beaker and add 5 mL of a mixture of 1:1 __ (CAUTION: HIGHLY FLAMMABLE. KEEP AWAY FROM OPEN FLAMES). Stir the casein in the ether for a few minutes, decant the ether, and repeat the process with a second 5 mL portion of ether. After the second washing with ether, filter the product. The ether washings remove any small quantities of fat that may have precipitated with the casein. Place the casein between several layers of pre weighed paper towels to help dry the product.

Answer 18

• powdered non-fat dry milk
• 1.7 M acetic acid
• ethyl ether and ethanol

Question 19

Specifics: Prodecure 3 (Exercise 4)

Place 0.1 g of __ into a 50 mL volumetric flask, add 25 mL distilled water (pre-warmed to 38˚C) and 5 mL of 1 M NaOH. Shake gently to dissolve the casein. Add 2.9 mL of 1.7 M CH3COOH, mix, cool, and fill up to 50 mL. Place 1 mL of this solution into three test tubes containing 10 mL __,10 mL __, and 10 mL __. Allow to stand for 30 minutes and observe for cloudiness.

Answer 19

• isolated casein
• glycine-HCl buffer (pH 2.7)
• acetate buffer (pH 4.7)
• phosphate buffer (pH 6.7)