Class notes

Biochemistry - Proteins

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Course
Bio 1000

Institution
University Of Manitoba (UManitoba )

Hi everyone ! These notes will definitely help you with Biochemistry. All the notes are precise and contain all the points you should know about these topics. So, you can use them as class notes, as well as summaries to get prepared well for your exams. Hope you guys will love these !

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Uploaded on June 30, 2022
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Biochemistry Exam Aid

CA$ 76.16 CA$ 79.14 6 items

1. Class notes - Biochemistry notes
2. Class notes - Biochemistry - enzymes
3. Class notes - Biochemistry - glycolysis
4. Class notes - Biochemistry - glycogenesis
5. Class notes - Biochemistry - proteins
6. Class notes - Biochemistry - tca cycle
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Exploring Proteins

(1) Purification of proteins
(2) Sequence analysis of proteins (sequencing)
(3) Immunological analysis of proteins
(4) Determination of 3-D structures of proteins

The Purification of Proteins Is an Essential First Step in Understanding Their Function

• Goal : to yield only one type of molecule
• Starting from pure proteins, we can determine amino acid sequences and investigate biochemical
functions.
• From the amino acid sequences, we can map evolutionary relationships between proteins in diverse
organisms .
• By using crystals grown from pure protein, we can obtain x-ray data that will provide us with a
picture of the protein’s tertiary structure— the shape that determines function.
• Purification should yield a sample containing only one type of molecule— the protein in which the
biochemist is interested.
• This protein sample may be only a fraction of 1% of the starting material, whether that starting
material consists of one type of cell in culture or a particular organ from a plant or animal.
• A protein can be purified by subjecting the impure mixture of the starting material to a series of
separations based on physical properties such as size and charge.
• To monitor the success of this purification, the biochemist needs a test, called an assay, for some
unique identifying property of the protein.

Protein must be released from the cell to be purified

• Source of protein, the starting material.
• Having found an assay and chosen a source of protein, we now fractionate the cell into components
and determine which component is enriched in the protein of interest.

,• In the first step, a homogenate is formed by disrupting the cell membrane, and the mixture is
fractionated by centrifugation, yielding a dense pellet of heavy material at the bottom of the
centrifuge tube and a lighter supernatant above
• The supernatant is again centrifuged at a greater force to yield yet another pellet and supernatant.
• This procedure, called differential centrifugation, yields several fractions of decreasing density, each
still containing hundreds of different proteins.
• The fractions are each separately assayed for the desired activity.
• Usually, one fraction will be enriched for such activity, and it then serves as the source of material to
which more-discriminating purification techniques are applied.

Proteins can be purified according to solubility, size, charge, and binding affinity

• Usually, protein mixtures are subjected to a series of separations, each based on a different
property.
• At each step in the purification, the preparation is assayed and its specific activity is determined.
• A variety of purification techniques are available.

, 1. Salting Out
• Most proteins are less soluble at high salt concentrations, an effect called salting out . The salt
concentration at which a protein precipitates differs from one protein to another. Hence, salting out
can be used to fractionate proteins.
• Salting out is also useful for concentrating dilute solutions of proteins, including active fractions
obtained from other purification steps.
• less soluble at high salt concentration to fractionate proteins
• ammonium sulfate, NaCl sample: 50% saturation of (NH4)2SO4 to precipitate globulins from serum
• Dialysis can be used to remove the salt if necessary
• Dialysis. Proteins can be separated from small molecules such as salt by dialysis through a
semipermeable membrane, such as a cellulose membrane with pores
• The protein mixture is placed inside the dialysis bag, which is then submerged in a buffer solution
that is devoid of the small molecules to be separated away.
• Molecules having dimensions significantly greater than the pore diameter are retained inside the
dialysis bag. Smaller molecules and ions capable of passing through the pores of the membrane
diffuse down their concentration gradients and emerge in the solution outside the bag.
• This technique is useful for removing a salt or other small molecule from a cell fractionate, but it will
not distinguish between proteins effectively .

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