Lecture 6 Protein purification techniques II Flashcards
Whats another way of separating proteins?
It is possible to separate proteins by differences in their surface electric charge
Whats the definition of pI?
“isoelectric point,” this is the pH at which a molecule has a net neutral charge.
What techniques exist to separate proteins based on their surface electric charge?
+ Isoelectric Focusing:
- On a pH gradient gel with an applied electric field, a protein will move within the field until the charge on a protein is zero -This is its Isoelectric Point (pI)
- Proteins can be separated this way
+ Ion-exchange Chromatography:
- Beads on a column with a given attached surface charge will bind proteins with an opposing charge
- The bound proteins are then separated and removed by the addition of a gradually increasing concentration of a salt
+ Gel Electrophoresis:
- Most common form of the technique is: SDS PAGE
- Separates proteins both by size and charge
- This technique can measure a proteins mass
+ Affinity Chromatography:
- Separates proteins with specific binding for a small molecule attached to beads on a column
- The protein is eluted off by passing a concentrated solution of the ‘free’ small molecule down the column
Describe the technique Isoelectric focusing.
- This technique requires a pH gradient gel
- It uses a gel of polyampholytes
- Polyampholytes are small multi-charged polymers with different values of pI
- Applying an electric field to this gel creates the pH gradient
- Proteins have differing numbers of acidic and basic residues
- They often have an overall positive or negative charge
- Proteins placed into the gel move within the applied electric field according to their surface charge
- When the surrounding pH equals the pI value for any given protein it will cease to move in the field
- It is isoelectrically focused
- A high resolution degree of separation can be obtained
Describe the technique Ion-exchange chromatography.
- Ion-exchange chromatography uses beads with a surface charge chemically attached to them
- This can be a negative (Carboxymethyl - CM) OR positive (Diethylaminoethyl - DEAE) charge
- The beads are typically cellulose or agarose
+ Example: Proteins with positive charge will attach to negatively charged beads - Other proteins will pass down the column unhindered
- The positively charged proteins can then be eluted from the column: (Eluting = Releasing)
- Eluting the bound proteins
- Add a low concentration of a salt + As an example: sodium chloride
- Sodium ions are clearly very strongly positive
- The sodium ions bind to the beads instead of the proteins
- Weakly positive proteins will elute off first
- Increasing the salt concentration will cause more positively charged proteins to elute from the column
- All the positively charged proteins can be collected as fractions as they come off the column= The other way round would work too
- Positive beads can be used to separate negatively
charged proteins
Describe the technique Gel-electropheresis
- Gel Electrophoresis separates proteins according to their size by applying an electric charge through a polymer gel
- A polyacrylamide gel is almost always used
- The technique is known as: Polyacrylamide Gel Electrophoresis ==> PAGE
- Polyacrylamide is chemically inert
- The gel forms as ‘SPAGHETTI-LIKE’ STRANDS
- The most common form of gel electrophoresis uses:
Sodium Dodecyl Sulphate ==> SDS= Hence SDS-PAGE - SDS is an anionic detergent
- It disrupts all non-covalent interactions in proteins
- SDS binds to amino acid residues in a ratio of around 1:2
- All proteins become negatively charged
- The negative charge on each protein becomes directly proportional to its mass
- β-Mercaptoethanol is added to disrupt disulphide bonds if there are any present
- The proteins are now FULLY DENATURED
- The protein mixture flows down the gel from the cathode towards the anode
- Large proteins are impeded in the gel by the strands
- The smaller proteins pass easily between the strands
Summarise:
Smallest proteins fastest through the Strands (SfS) - The protein separation provides a direct measurement of their masses
- Proteins with known molecular masses are run as a
scale marker beside those with unknown mass - Differences in mass of ~2% between proteins can be
determined using SDS-PAGE - Around 10 residues difference
Describe the technique Affinity Chromatography.
- Affinity Chromatography makes use of the fact that many proteins tightly bind small specific molecules as part of their function
Example: Concanavalin A - Concanavalin A binds glucose very tightly
- It is possible to covalently attach glucose to beads in a column
- Pass the crude protein mixture containing Concanavalin A down the column
- The Concanavalin A will bind to the glucose on the beads
- All remaining proteins will pass through unhindered
- Concanavalin A can then be removed by passing a concentrated solution of glucose down the column
- Concanavalin A binds better to the ‘free’ glucose than to the ‘bound’ glucose on the beads
- The Concanavalin A will elute from the column bound to the ‘free’ glucose
- Typically the ‘free’ glucose would be removed by dialysis
What is the general procedure all of these techniques follow?
- A protein, Y, recognises and binds a small molecule, X
- Covalently attach X to beads and place these in a column
- In a mixture of proteins containing Y only that protein will bind to X on the beads and will be retained by the column
- The rest of the proteins will pass on through the column
- Passing down the column a concentrated solution of ‘free’ X will remove protein Y from the column
- Protein Y will be removed as pure protein
