volume to load sample for affinity chromatography - (Feb/10/2011 )

Hi,

I have a question regarding about how much the volume of sample that can be loaded to run chromatography. I'm using HiTrap Chelating HP, 5ml to purify protein of interest.
Does it has any limit to load the sample? This are the details about the column:

Column volumes: 5ml
Column dimensions idxh: 1.6 x 2.5 cm
Chelating group: Iminodiacetic acid
Binding capacity: ~12 mg pure (histidine)6-tagged protein (Mr, ~27 600) per ml/medium

I've run several chromatography before and I loaded all the sample in one shot. I never thought that I need to calculate how much sample that can be loaded at one time.
Is it true? My supervisor scolded me because of that and I'm afraid to ask him the calculation. Please help me.
And thank you very much for any information or suggestion.

-rpjkmust916-

rpjkmust916 on Thu Feb 10 11:30:36 2011 said:

Hola I don´t understand why your supervisor questions your work, because you are right (at least working with lab scale cultures. You have total capacity to bound around 6o mg of pure protein of a mol. weight medium (30-50) KD as yours. How % of total protein is yours ? 10% .- You could pass 600mg of total protein each run. 5% ----> about 1.2g. 1%? ----> about 6g. So measure the protein conc. of your extrac and made an stimation of the % of recombinant protein against total protein and calculate how many parts of each lot you have pass through the colum. Buena suerte

-protolder-

Hi Protolder,

Thanks for your reply and suggestion. The truth is, I'm new in molecular biology and also in protein purification. I've just follow what my senior taught me. I've never measured the total protein concentration before I load it to the column. I've only measured after purified and concentrated it. That's also one thing that made him very angry with me that I've never measured the total protein concentration. Could you explain when I should measure the protein concentration?

And thank you very much for your reply.

-rpjkmust916-

Total protein concentration you can work out with a protein assay such as bradford or lowry. If you run your sample on an SDS-PAGE and stain it with Coomassie you can work out roughly how much your recombinant protein is in terms of % of total protein. Or if you need to work it out exactly, perhaps there are bio-assays/ELISA kits available that let you quantify protein based on activity.

A quicker way than lowry/bradford is to just nanodrop the sample. This however will be much less accurate because you cannot account for extinction co-efficients if your protein prep is not homogeneous.

EDIT: In terms of when to do it, I'd use samples from each significant stage of purification. I assume you are using bacteria to express protein? If so, test total protein from your lysate, soluble fraction, filtered fraction, HiTrap FT and HiTrap elute. That's what I'd do anyway.

-SebS-

Hi SebS,

Thank you very much for your reply.
Yes, the expression of the protein was done with E.coli. Usually I run SDS-Page to see wheteher the fraction collected has the purified protein or not. And after that I will pool all the tubes with the fraction and concentrated it with Vivaspin tube. And finally measured the protein concentration with Bradford assay.
May I ask, what do you mean by soluble fraction and filtered fraction. I run the SDS-Page with the Lysate, HiTrap FT, Wash fraction and HiTrap elute.
Hope my basic question won't make you mad.
Thanks in advance.
Any suggestions from everybody are welcome.

-rpjkmust916-

rpjkmust916 on Fri Feb 11 08:32:06 2011 said:

Hola, sorry by my intromission. SebS, refers to soluble fraction at the supernatant of centrifugation of the lysated culture, but before load the column is adequate filter it at least by 0.8 or 0.45 in order to avoid blocking the column. Buena suerte

-protolder-

Hi Protolder,

Thank you very much for the explanation !!!

-rpjkmust916-