Slide 1Overview
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Reasons for lysis
Soluble proteins made by cell, including red fluorescent protein, are dissolved in cell cytoplasm
Only way to access soluble proteins is to lyse (break open) cell
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Obtain 1mL cell culture in tube
Centrifuge for 5 min and identify location of red fluorescent protein
Remove supernatant with pipette
Add another 1mL of cell culture to tube and centrifuge for 5 min
Remove supernatant with pipette
Procedure
Tip tube and remove last bit of supernatant with pipette, without touching cell pellet
Add elution buffer and drag closed tube across tube rack to resuspend cells
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Reasons for separation
Although the bacteria make a lot of red fluorescent protein, there are up to 1,000 other proteins in a living cell
Those other proteins might interfere with intended use of RFP or of any other protein you are isolating
Pharmaceutical companies require purified protein
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Procedure
Centrifuge cells (be sure to balance), and remove supernatant with RFP
Add binding buffer to the supernatant
Add supernatant to column and drain
Add wash buffer to column and drain
Add elution buffer to column and collect RFP
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Never touch the resin with pipette
Drain until 2 mm of liquid is left above the column and never expose the resin to air
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Difference is electric charge distribution
Hydrophobic amino acids ARE NOT attracted to water molecules
Hydrophilic proteins ARE attracted to water molecules
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In binding buffer, hydrophobic proteins unfold
Unfolded hydrophobic proteins adhere to the hydrophobic column resin
Folded hydrophilic proteins never adhere to the column
Hydrophilic proteins
In wash buffer, moderately hydrophobic proteins fold
Highly hydrophobic proteins, including RFP, stay unfolded
Folded moderately hydrophobic proteins are released from the column
Moderately hydrophobic proteins
In elution buffer, highly hydrophobic proteins, including RFP, fold
Folded highly hydrophobic proteins, including RFP, are released from the column
RFP can be collected
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E–6 CHAPTER 6 | STUDENT GUIDE © 2013 Amgen Foundation. All rights reserved.
When a protein is first synthesized, it is a long flexible chain of amino acids, but it immediately attains its three-dimensional conformation by a process called protein folding (see Figure 6.2). Protein folding is dependent on the following properties of the amino acids in the protein:
• Formation of weak noncovalent bonds between positively charged and negatively charged side chains of amino acids
• The tendency for hydrophobic (water-insoluble) amino acids to be buried on the inside of the protein away from water and for hydrophilic (water- soluble) amino acids to be found on the outside of the protein exposed to water
• Formation of covalent bonds, called disulfide bridges, that occur between sulfur-containing amino acids
Figure 6.2: Protein folding
CONSIDER: If a mutation changes an amino acid, how might this change affect protein folding and protein function?
Specific proteins are either hydrophobic or hydrophilic, depending on the relative amount of hydrophobic and hydrophilic amino acids they contain. Hydrophobic proteins and hydrophilic proteins can be separated by column chromatography. In this method, a column is packed with small beads that are coated with a material (a resin) that attracts hydrophobic amino acids, and the mixture of proteins is dissolved and passed over the beads. In order for the hydrophobic amino acids to stick to the resin, the proteins must be unfolded to expose the hydrophobic amino acids, which tend to be found on the inside of the protein. Certain salt solutions called buffers will unfold proteins to varying degrees.
Unfolded Folded
Resin-coated beads
! ! !
!
E–6 CHAPTER 6 | STUDENT GUIDE © 2013 Amgen Foundation. All rights reserved.
When a protein is first synthesized, it is a long flexible chain of amino acids, but it immediately attains its three-dimensional conformation by a process called protein folding (see Figure 6.2). Protein folding is dependent on the following properties of the amino acids in the protein:
• Formation of weak noncovalent bonds between positively charged and negatively charged side chains of amino acids
• The tendency for hydrophobic (water-insoluble) amino acids to be buried on the inside of the protein away from water and for hydrophilic (water- soluble) amino acids to be found on the outside of the protein exposed to water
• Formation of covalent bonds, called disulfide bridges, that occur between sulfur-containing amino acids
Figure 6.2: Protein folding
CONSIDER: If a mutation changes an amino acid, how might this change affect protein folding and protein function?
Specific proteins are either hydrophobic or hydrophilic, depending on the relative amount of hydrophobic and hydrophilic amino acids they contain. Hydrophobic proteins and hydrophilic proteins can be separated by column chromatography. In this method, a column is packed with small beads that are coated with a material (a resin) that attracts hydrophobic amino acids, and the mixture of proteins is dissolved and passed over the beads. In order for the hydrophobic amino acids to stick to the resin, the proteins must be unfolded to expose the hydrophobic amino acids, which tend to be found on the inside of the protein. Certain salt solutions called buffers will unfold proteins to varying degrees.
Unfolded Folded
Resin-coated beads
!
E–6 CHAPTER 6 | STUDENT GUIDE © 2013 Amgen Foundation. All rights reserved.
When a protein is first synthesized, it is a long flexible chain of amino acids, but it immediately attains its three-dimensional conformation by a process called protein folding (see Figure 6.2). Protein folding is dependent on the following properties of the amino acids in the protein:
• Formation of weak noncovalent bonds between positively charged and negatively charged side chains of amino acids
• The tendency for hydrophobic (water-insoluble) amino acids to be buried on the inside of the protein away from water and for hydrophilic (water- soluble) amino acids to be found on the outside of the protein exposed to water
• Formation of covalent bonds, called disulfide bridges, that occur between sulfur-containing amino acids
Figure 6.2: Protein folding
CONSIDER: If a mutation changes an amino acid, how might this change affect protein folding and protein function?
Specific proteins are either hydrophobic or hydrophilic, depending on the relative amount of hydrophobic and hydrophilic amino acids they contain. Hydrophobic proteins and hydrophilic proteins can be separated by column chromatography. In this method, a column is packed with small beads that are coated with a material (a resin) that attracts hydrophobic amino acids, and the mixture of proteins is dissolved and passed over the beads. In order for the hydrophobic amino acids to stick to the resin, the proteins must be unfolded to expose the hydrophobic amino acids, which tend to be found on the inside of the protein. Certain salt solutions called buffers will unfold proteins to varying degrees.
Unfolded Folded
Resin-coated beads
!
E–6 CHAPTER 6 | STUDENT GUIDE © 2013 Amgen Foundation. All rights reserved.
When a protein is first synthesized, it is a long flexible chain of amino acids, but it immediately attains its three-dimensional conformation by a process called protein folding (see Figure 6.2). Protein folding is dependent on the following properties of the amino acids in the protein:
• Formation of weak noncovalent bonds between positively charged and negatively charged side chains of amino acids
• The tendency for hydrophobic (water-insoluble) amino acids to be buried on the inside of the protein away from water and for hydrophilic (water- soluble) amino acids to be found on the outside of the protein exposed to water
• Formation of covalent bonds, called disulfide bridges, that occur between sulfur-containing amino acids
Figure 6.2: Protein folding
CONSIDER: If a mutation changes an amino acid, how might this change affect protein folding and protein function?
Specific proteins are either hydrophobic or hydrophilic, depending on the relative amount of hydrophobic and hydrophilic amino acids they contain. Hydrophobic proteins and hydrophilic proteins can be separated by column chromatography. In this method, a column is packed with small beads that are coated with a material (a resin) that attracts hydrophobic amino acids, and the mixture of proteins is dissolved and passed over the beads. In order for the hydrophobic amino acids to stick to the resin, the proteins must be unfolded to expose the hydrophobic amino acids, which tend to be found on the inside of the protein. Certain salt solutions called buffers will unfold proteins to varying degrees.
Unfolded Folded
Resin-coated beads
© 2013 Amgen Foundation. All rights reserved.
When a protein is rst synthesized, it is a long exible chain of amino acids, but it immediately attains its three-dimensional conformation by a process called protein folding (see Figure 6.2). Protein folding is dependent on the following properties of the amino acids in the protein:• Formation of weak noncovalent bonds between positively charged and negatively charged side chains of amino acids • The tendency for hydrophobic (water-insoluble) amino acids to be buried on the inside of the protein away from water and for hydrophilic (water-soluble) amino acids to be found on the outside of the protein exposed to water• Formation of covalent bonds, called disulde bridges, that occur between sulfur-containing amino acids Figure 6.2: Protein foldingCONSIDER: If a mutation changes an amino acid, how might this change affect protein folding and protein function?Specic proteins are either hydrophobic or hydrophilic,
depending on the relative amount of hydrophobic and
hydrophilic amino acids they contain. Hydrophobic
proteins and hydrophilic proteins can be separated by
column chromatography. In this method, a column is
packed with small beads that are coated with a material
(a resin) that attracts hydrophobic amino acids, and the
mixture of proteins is dissolved and passed over the
beads. In order for the hydrophobic amino acids to stick
to the resin, the proteins must be unfolded to expose the
hydrophobic amino acids, which tend to be found on the
inside of the protein. Certain salt solutions called buffers
will unfold proteins to varying degrees.
UnfoldedFoldedResin-coated
beads
Column
© 2013 Amgen Foundation. All rights reserved.
When a protein is rst synthesized, it is a long exible chain of amino acids, but it immediately attains its three-dimensional conformation by a process called protein folding (see Figure 6.2). Protein folding is dependent on the following properties of the amino acids in the protein:• Formation of weak noncovalent bonds between positively charged and negatively charged side chains of amino acids • The tendency for hydrophobic (water-insoluble) amino acids to be buried on the inside of the protein away from water and for hydrophilic (water-soluble) amino acids to be found on the outside of the protein exposed to water• Formation of covalent bonds, called disulde bridges, that occur between sulfur-containing amino acids Figure 6.2: Protein foldingCONSIDER: If a mutation changes an amino acid, how might this change affect protein folding and protein function?Specic proteins are either hydrophobic or hydrophilic,
depending on the relative amount of hydrophobic and
hydrophilic amino acids they contain. Hydrophobic
proteins and hydrophilic proteins can be separated by
column chromatography. In this method, a column is
packed with small beads that are coated with a material
(a resin) that attracts hydrophobic amino acids, and the
mixture of proteins is dissolved and passed over the
beads. In order for the hydrophobic amino acids to stick
to the resin, the proteins must be unfolded to expose the
hydrophobic amino acids, which tend to be found on the
inside of the protein. Certain salt solutions called buffers
will unfold proteins to varying degrees.
UnfoldedFoldedResin-coated
beads
Column
© 2013 Amgen Foundation. All rights reserved.
When a protein is rst synthesized, it is a long exible chain of amino acids, but it immediately attains its three-dimensional conformation by a process called protein folding (see Figure 6.2 ). Protein folding is dependent on the following properties of the amino acids in the protein:• Formation of weak noncovalent bonds between positively charged and negatively charged side chains of amino acids • The tendency for hydrophobic (water-insoluble) amino acids to be buried on the inside of the protein away from water and for hydrophilic (water-soluble) amino acids to be found on the outside of the protein exposed to water• Formation of covalent bonds, called disulde bridges, that occur between sulfur-containing amino acids Figur e 6.2: Pr otein foldingCONSIDER: If a mutation changes an amino acid, how might this change affect protein folding and protein function?Specic proteins are either hydrophobic or hydrophilic,
depending on the relative amount of hydrophobic and
hydrophilic amino acids they contain. Hydrophobic
proteins and hydrophilic proteins can be separated by
column chromatography. In this method, a column is
packed with small beads that are coated with a material
(a resin) that attracts hydrophobic amino acids, and the
mixture of proteins is dissolved and passed over the
beads. In order for the hydrophobic amino acids to stick
to the resin, the proteins must be unfolded to expose the
hydrophobic amino acids, which tend to be found on the
inside of the protein. Certain salt solutions called buffers
will unfold proteins to varying degrees.
UnfoldedFoldedResin-coated
beads
Column