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Predicting amount maximum of sample to apply onto cation exchange chromatography


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#1 trungnghiatn

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Posted 21 April 2015 - 01:14 AM

Hi,

 

I'm calculating amount of sample to apply onto cation exchange chromatography column.

 

My protein is PDGF (30kDa), which have 24e of net charged. I used Hitrap SP FF column 5ml to purify. In the user guide of GE, they show that the column's capacity is 70mg Ribonuclease A (13.7kDa). I found that the net charged of Ribonuclease A is 29e.

 

We have a table:

hRss7ML.png?1

 

I calculated that: The total charge between two samples is equal. Therefore, x= (70/13.7*29)/24*30 = 179.1 (mg)

 

I don't know what I do right or wrong? Please help me. Thanks,

 

* Calculating total charge of protein here: http://nbcr-222.ucsd.../pdb2pqr_2.0.0/

** Getting PDB ID here: http://www.rcsb.org/pdb/home/home.do


Edited by trungnghiatn, 21 April 2015 - 01:16 AM.


#2 labtastic

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Posted 21 April 2015 - 04:30 PM

There really isn't any reliable means to predict binding capacity with your protein.

 

Capacity is determined by more factors than just molecular weight and net charge. Protein shape, oligomerization state, post-translational modification status, buffer composition, and surface charge density distribution, to name a few, are important factors that will influence binding capacity.

 

The reported binding capacities are meant as a guideline. Best way to know for sure is saturate your resin with your protein and see how much elutes. Or just start conservative (10mg/ml resin) and work your way up.



#3 trungnghiatn

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Posted 21 April 2015 - 07:07 PM

There really isn't any reliable means to predict binding capacity with your protein.

 

Capacity is determined by more factors than just molecular weight and net charge. Protein shape, oligomerization state, post-translational modification status, buffer composition, and surface charge density distribution, to name a few, are important factors that will influence binding capacity.

 

The reported binding capacities are meant as a guideline. Best way to know for sure is saturate your resin with your protein and see how much elutes. Or just start conservative (10mg/ml resin) and work your way up.

 

I wonder how can I configure out amount of PDGF which can be applied. It will be a basis for me to design experiment. I don't think I can use this result like a reference.



#4 labtastic

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Posted 22 April 2015 - 10:43 AM

I think you misunderstood my post. You cannot predict how much protein will bind to your resin. There are too many factors.

 

Here is how to figure out how much PDGF will bind to your resin:

 

1. Saturate a small 1 ml column with your protein (this can be done by loading protein until no more binds, e.g. with an excess of cell lysate from a large [multiple liters] over expression)

2. Wash resin

3. Elute protein from resin

4. Quantitate the amount of eluted protein

 

Then do your experiment.



#5 trungnghiatn

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Posted 23 April 2015 - 05:20 AM

I think you misunderstood my post. You cannot predict how much protein will bind to your resin. There are too many factors.

 

Here is how to figure out how much PDGF will bind to your resin:

 

1. Saturate a small 1 ml column with your protein (this can be done by loading protein until no more binds, e.g. with an excess of cell lysate from a large [multiple liters] over expression)

2. Wash resin

3. Elute protein from resin

4. Quantitate the amount of eluted protein

 

Then do your experiment.

 

I have tried with an old column. At that time, I applied more 350ml sample without delution. I don't have a new column which have same size with the old column but I'm going to test in larger column. Therefore, I want predict for next experiments to save the sample. So hard to have a large of sample.



#6 mdfenko

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Posted 24 April 2015 - 04:00 AM

you should be able to recover any unbound protein which can be reapplied after eluting the initially bound protein. there is, effectively, no loss of sample.


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