From my own experience, I know that when you are an undergraduate student and you have to just study about all different techniques without trying them, it is hard to comprehend anything. So I decided to create the GGS LIVE section in which I will try to bring closer as much techniques as possible by showing a real stuff (solving real problems) from the lab. This is first time I do such a thing so please be understanding:) It will get better:)
Today....
METHOD: Protein purification.
ABOUT: What is a protein purification process? In my opinion it is isolation of the specific protein out of the complex protein mixture or an enrichment of it in the outcome sample. Properties of the target protein always force the way of its purification. Simply speaking if the target protein A is a negatively charged we would use a positively charged substance X to pull it down from the mixture.
WHAT: Purification of the IgG (immunoglobulin G) fraction from rabbit serum.
HOW: Protein A - is an bacterial protein that efficiently interacts with IgG's.
METHOD SCHEME:
ANALYSIS: On each step of the purification, protein samples are collected which then are separated by a PAGE (PolyAcrylamind Gel Ecectrophoresis). After that proteins are visualized by staining with Commasie dye (Brilliant blue).
What you see here is a Coomasie gel where proteins are visualized by staining with blue dye. Simply gel after electrphopresis is incubated with the dye solution and then gel is destained (gel gets stained where there is no proteins as well).
Before we start I have to tell you that IgG's like other antibodies are build up of two different sets of chains, called heavy and light chain which molecular sizes are around 50kDa and 25kDa, respectively.
Let's start then!! Simply lanes load and unbound represent a starting material (crude serum) and sample after binding, respectively. You can see that there is not much difference in proteins content between those two samples. Why is that? Because only small amount of the beads was used in this particular experiment (this is a pilot experiment:). Simply there is still so much of the protein left after binding that we do not see the difference (if greater amount of the beads was used we would see depletion of the protein in the unbound sample)
Lanes Wash 1 - 4 are simply a samples that contains proteins that did not bind to the beads. You can see that there is some of protein of interest in these samples as well. This also suggests that we beads were satureted.
.
Lane Beads is the sample that represents what was present on the beads before the elution step. You can see that a good amount of "our" protein did stick to the beads and was not washed away. Additionally you can see that most of the contamitants present in the load sample is not present in this one.
Lanes Elution 1 - 5 represents samples recovered from the beads. Again there is a good amount of our protein in thsese samples (I do not know what happend in the elution 3:). You can see that most of the protein did come off the column in two first elutions.
Lane Beads after elution represents ... I do not have to tell ya:) You can see that there is still some amount of "our" protein present on the beads which can be eluted (it was eluted but I have in on a different gel).
Last lane represents sample which simply is a pull of all fractions which then were concentrated. So this sample is our final one. You can see that we have both chains present (which is good:) and that there is not much of the contamitants.
Simply we have managed to purify the IgG's fraction from the rabbit serum.
In the next part: CLONING
CyA!
Maciek
RESULTS:
What you see here is a Coomasie gel where proteins are visualized by staining with blue dye. Simply gel after electrphopresis is incubated with the dye solution and then gel is destained (gel gets stained where there is no proteins as well).
Before we start I have to tell you that IgG's like other antibodies are build up of two different sets of chains, called heavy and light chain which molecular sizes are around 50kDa and 25kDa, respectively.
Let's start then!! Simply lanes load and unbound represent a starting material (crude serum) and sample after binding, respectively. You can see that there is not much difference in proteins content between those two samples. Why is that? Because only small amount of the beads was used in this particular experiment (this is a pilot experiment:). Simply there is still so much of the protein left after binding that we do not see the difference (if greater amount of the beads was used we would see depletion of the protein in the unbound sample)
Lanes Wash 1 - 4 are simply a samples that contains proteins that did not bind to the beads. You can see that there is some of protein of interest in these samples as well. This also suggests that we beads were satureted.
.
Lane Beads is the sample that represents what was present on the beads before the elution step. You can see that a good amount of "our" protein did stick to the beads and was not washed away. Additionally you can see that most of the contamitants present in the load sample is not present in this one.
Lanes Elution 1 - 5 represents samples recovered from the beads. Again there is a good amount of our protein in thsese samples (I do not know what happend in the elution 3:). You can see that most of the protein did come off the column in two first elutions.
Lane Beads after elution represents ... I do not have to tell ya:) You can see that there is still some amount of "our" protein present on the beads which can be eluted (it was eluted but I have in on a different gel).
Last lane represents sample which simply is a pull of all fractions which then were concentrated. So this sample is our final one. You can see that we have both chains present (which is good:) and that there is not much of the contamitants.
Simply we have managed to purify the IgG's fraction from the rabbit serum.
In the next part: CLONING
CyA!
Maciek
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