This is very eays to follow and makes sense. Keeping the column hydrated to allow the Hb-Catalase elution to remain consistent, adding in drops to ensure the flow through is not too fast to capture, and keeping an eye on the orange-brown substance that demarcates the protein soltuion being taken. It's so carefully shown and described that you couldn't fail to replciate it. Marvellous. What I cannot make sense of is the last 2 minutes. Are you adding in the protein solution between to rounds of the phosphate buffer, rather than in the previous steps where the solution was added before the buffer each time? It seems as if the protein is added over a little phophate buffer, then eluted out under another load of buffer. Is Haemoglobin then measured by mixing the soltuions with a solution of 2.08mmol/L of SLS (Sodium lauryl sulphate) and reading absorbance at 539nm?
No. The protein mix is loaded on at the start. After the first protein is eluted, I then switch to a different buffer to elute the second protein that is bound to the column. And, no, I did not use a spec. That is, I did not measure the amount of haemoglobin eluted.
@@NickMorrisPhD Fair play for answering so fast. I'm in the throes of learning this technique at school now and I really wish to know the underpinnings and hardcore basics of it down to the tee. Can one learn of the purposes of the different buffers?
@@owenhunt The buffers are essentially the same, apart from the concentration of phosphate. Both buffers are pH6.5 and the first buffer contains 17.5 mM phosphate and the second 175 mM phosphate. The 17.5 mM is used first and at this concentration of phosphate the catalase will bind to the column and the haemoglobin will not, and so the haemoglobin flows through to be collected. When we switch to the 175 mM buffer, the phosphate ions compete with the catalase for binding to the column. This results in the catalase becoming unbound and so it is eluted.
According to my notes there were 0.88 g of haemoglobin per 100 ml, and haemoglobin and 10 mg of catalase. However, these were our starting numbers and we often had to change them due to batch variation in supplied purified proteins.
swanand deshmukh - the resin is an issue, as the one used - DEAE-Sepharose - is either no longer made, or is in short supply (or it was a couple of years ago when I tried to buy some). The sepharose needs washing as outlined in the manufacturer’s instructions to activate it, and then it is poured just as you would pour any column. You get the sepharose in suspension and pour it in one go into the column. Whether or not you leave the tap on at the bottom of the column is up to you. Letting the liquid flow out can result in quicker packing, but a ‘tight’ column with restricted flow rates (there is also the risk of the column running dry), whereas leaving the sepharose to settle can take a lot longer, can give better flow rates, and reduces the risk of the column running dry as it packs.
This is very eays to follow and makes sense. Keeping the column hydrated to allow the Hb-Catalase elution to remain consistent, adding in drops to ensure the flow through is not too fast to capture, and keeping an eye on the orange-brown substance that demarcates the protein soltuion being taken. It's so carefully shown and described that you couldn't fail to replciate it. Marvellous.
What I cannot make sense of is the last 2 minutes. Are you adding in the protein solution between to rounds of the phosphate buffer, rather than in the previous steps where the solution was added before the buffer each time?
It seems as if the protein is added over a little phophate buffer, then eluted out under another load of buffer.
Is Haemoglobin then measured by mixing the soltuions with a solution of 2.08mmol/L of SLS (Sodium lauryl sulphate) and reading absorbance at 539nm?
No. The protein mix is loaded on at the start. After the first protein is eluted, I then switch to a different buffer to elute the second protein that is bound to the column. And, no, I did not use a spec. That is, I did not measure the amount of haemoglobin eluted.
@@NickMorrisPhD Fair play for answering so fast. I'm in the throes of learning this technique at school now and I really wish to know the underpinnings and hardcore basics of it down to the tee.
Can one learn of the purposes of the different buffers?
@@owenhunt The buffers are essentially the same, apart from the concentration of phosphate. Both buffers are pH6.5 and the first buffer contains 17.5 mM phosphate and the second 175 mM phosphate. The 17.5 mM is used first and at this concentration of phosphate the catalase will bind to the column and the haemoglobin will not, and so the haemoglobin flows through to be collected. When we switch to the 175 mM buffer, the phosphate ions compete with the catalase for binding to the column. This results in the catalase becoming unbound and so it is eluted.
Hello Dr. Morris- Can you say how much of each protein was present in the 200 microlitres that was added at the beginning?
According to my notes there were 0.88 g of haemoglobin per 100 ml, and haemoglobin and 10 mg of catalase. However, these were our starting numbers and we often had to change them due to batch variation in supplied purified proteins.
How to fill own coloum..? Where to finf resin
swanand deshmukh - the resin is an issue, as the one used - DEAE-Sepharose - is either no longer made, or is in short supply (or it was a couple of years ago when I tried to buy some). The sepharose needs washing as outlined in the manufacturer’s instructions to activate it, and then it is poured just as you would pour any column. You get the sepharose in suspension and pour it in one go into the column. Whether or not you leave the tap on at the bottom of the column is up to you. Letting the liquid flow out can result in quicker packing, but a ‘tight’ column with restricted flow rates (there is also the risk of the column running dry), whereas leaving the sepharose to settle can take a lot longer, can give better flow rates, and reduces the risk of the column running dry as it packs.
@@NickMorrisPhD i want to seperate and purify the serine protease from my crude extract...how can i do it...more easily and economically..?
@@swananddeshmukh9839 Sorry, no idea. Have you tried searching PubMed to see how other researchers have done it?
@@NickMorrisPhD i tried to search ...but each method is laboreious and much expensive
@@swananddeshmukh9839 Welcome to the world of science! I am still waiting to find my cheap and easy experiment.
This must be size exclusion rather than Ion exchange
No, separation is based on the different isoelectric points
No. It is as advertised. It's anion exchange.
Nope, it is anion exchange. The haemoglobin carries no charge so goes straight through the column.
At the pH the experiment is run haemoglobin has no change and so does not stick to the column, whereas the catalase carries a charge and sticks.