3 Molecular WeightThe lab in week 6 and 7 will involve separating a protein mixture by molecular weight using 2 methods: gel filtration and SDS PAGEGel filtration separates by the native molecular weightSDS PAGE separates by the subunit molecular weight
4 Gel FiltrationThis method relies on a column of beads of a specified pore size. This is known as a molecular sieve.Proteins (and other macromolecules) above a certain cut-off size cannot fit into the pores and so migrate down the outside of the beads. They will elute first.Smaller molecules below the cut-off can permeate the pores and so take longer to travel down the column.
5 An elution profileGel-filtration of the protein mixture. 1.2 ml of protein mixture (10 mg/ml) was loaded onto a 25 cm X 2.5 cm diam. Sephadex G-50 column equilibrated with buffer (50 mM Tris HCl, pH 7.5). The column was eluted with buffer at ~1 ml/min, collecting 2.5 ml fractions. The absorbance of each fraction was measured at 280 nm and 410 nm.LighterHeavier
6 SDS PAGEThis technique involves loading a sample of your mixture onto a polyacrylamide gel (PAGE). Polyacrylamide works like agarose except the matrix has smaller pores and so polyacrylamide gels separate smaller molecules (like proteins). Agarose is used for much larger molecules such as DNA and RNA.
7 SDS PAGEUnlike DNA and RNA proteins do not have a nice constant charge to mass ratio and can have any charge at a given pH, depending on their sequence, hence pI.To overcome this problem proteins are coated with a detergent, SDS, which makes them negatively charged.They then separate by molecular weight.
8 SDS PAGE They then separate by molecular weight. The SDS will disrupt the secondary, tertiary and quaternary structure so the subunits will separate. For this reason SDS-PAGE separates by subunit molecular weight.
10 Your TaskEach pair will be given a mixture of three proteins. This mixture will be unique to your group. Your mixture will contain between 4 and 8 mg of any three of the following proteins: Myoglobin, Haemoglobin, Cytochrome c, a-lactalbumin, Ribonuclease, Bovine Serum Albumin, Ferritin and Catalase. It is your task to separate and identify these three proteins.
11 Clues to help youYour mixture of three proteins will either contain 2 heavy and 1 light protein or 1 heavy and 2 light proteins. For the purposes of this experiment, a heavy protein is defined as one with a molecular weight of over 50,000 and a light protein is one with a molecular weight of less than 50,000. The two smaller/heavier proteins in the mixture have pI’s that differ by at least 2 pH units. These can be separated by ion exchange chromatography at pH 7.5.
12 Hint…..If you can separate 2 proteins by ion exchange chromatography at pH 7.5 then the 2 proteins must have pIs on either side of 7.5 so they are opposite charges at PpH 7.5.Knowing information about the possible proteins at the back of the notes for this lab session what can you conclude before coming to class?
13 Charge Ion Exchange Chromatography Native gel electrophoresis Isoelectric focusing
14 Charges on proteins Different proteins have different native charges. The overall charge on a protein will depend on:The sequenceThe pH
15 Determining the pI of a protein It can be predicted from the difference between the sum of the acidic side chains (asp + glu) and the sum of the basic side chains (lys + arg + his).It is determined experimentally by techniques such as isoelectric focusing. The protein is placed in a pH gradient and subjected to an electric field. The protein moves to its pI.
16 Determining the pI of a protein Those proteins with more acidic residues will have a lower pIThose proteins with more basic residues will have a higher pI.
17 Estimating the charge of a protein What we really want to know is the charge of a protein at a particular pH, like 7.How do we use pI data to predict the charge of our protein?Acidic residues lower the pIBasic residues raise the pI.
18 Estimating the charge of a protein pI ~5[H+][OH-][OH-][H+]Protein becomes increasingly +veProtein becomes increasingly -ve
19 Estimating the charge of a protein At pH 3 the protein will be +vepH ~3pI ~5[H+][OH-][OH-][H+]Protein becomes increasingly +veProtein becomes increasingly -ve
20 Estimating the charge of a protein At pH 7 the protein will be -vepI ~5[H+]pH ~7[OH-][OH-][H+]Protein becomes increasingly +veProtein becomes increasingly -ve
21 At a particular pH..If the pH of the environment is below (more acidic >[H+]) the pI then the protein will be positive (+ve)If the pH of the environment is above (more basic >[OH-]) the pI then the protein will be negative (-ve).
22 Ion Exchange Chromatography If the column is positively charged i.e. DEAE then….Proteins with pIs l < the pH of the buffer will be negatively charged and bind to the column.Proteins with pIs > the pH of the buffer will be positively charged and will not bind to the column but elute.
23 Ion Exchange Chromatography If the column is negatively charged charged i.e. carboxymethyl then….Proteins with pIs l < the pH of the buffer will be negatively charged and not bind to the column but elute.Proteins with pIs > the pH of the buffer will be positively charged and will bind to the column.
24 Native Gel Electrophoresis Proteins with pIs l < the pH of the buffer will be negatively charged and will move to the anode (+ve), the red electrode!!Proteins with pIs > the pH of the buffer will be positively charged and will move to the cathode (-ve), the black electrode!!
25 Isoelectric FocusingA pH gradient is set up along the length of the gelAn electric field is appliedProteins move to the point where they no longer have a charge i.e. their pIUsed as the first dimension of 2D gel electrophoresis
26 ProteomicsA combination of isoelectric focusing (first dimension) and SDS PAGE (second dimension) can separate the complete proteome of a cell!You produce spots which can be cut out and analysed by mass spectrometry.Compare to libraries of proteins
27 2D gel electrophoresisSDS PAGELow mol. Wt.High mol. Wt.pH310
28 Affinity Exploited with cloning His-tagged proteins purified on Nickel columns.GST fusion proteins purified on glutathione columns.