12 replies to this topic

[mirc]

Hi folks,
I´m an absolute beginner with regard to protein purification, but want to do some traditional protein clean-up (Anion/Cation EX; Gelfiltration).
I´m having tremendous problems with the first step: an anionexchange column (1ml Q XL from GE).
Whenever I load a cell lysate sample (~20mg, 2ml sample volume) nearly everything is in the flow through fraction and nothing really binds to the column as judged from WB and the chromatogram (I run: 1. 6ml 50mM NaCl buffer, then 15ml with a gradient form 50-1000mM NaCl; flow 0,5ml/min).
I don´t want to go below 50mM NaCl, cause I´m actually interested in a complex my protein of interest forms. But regardless of the 50mM more proteins should bind to the column initially...
Before adding the lysate on the column, the lysis buffer (150mM NaCl, 0,25% Deoxycholate, 1%NP40, etc, no SDS ) is exchanged to start buffer with a desalting column ( also tried dialysis).
I´ve also tried Glycerol in the buffers with the same result.

Can anyone give me a hint how to get my proteins to bind???

Thank you very much in advance!

post also posted in "Biochemistry"

Edited by mirc, 27 May 2010 - 03:52 AM.

[Inmost sun]

you may think of starting with hydrophobic interaction chromatography where you can start with high salt concentartions; my experience is that Q-columns are better at a late step when you have gotten rid off of bulk proteins...

which buffer and pH do you use?

[paramyosin]

Something is wrong, the mayority of the E. coli proteins (i assume is E. coli if not don´t read the following) are acidic, therefore they should bind to the column at pH of 7 (which pH you use in your buffer?). I think you are using too much extract just for 1 ml column, why don´t you use 200-500 ul of seed?. Are you analyzing your elution fractions by a SDS-PAGE? if you are controling just your FT probably you wouldn´t see nothing because you have too much protein. What do you use for your WB?, a monoclonal Ab against the protein or against a tag?. Maybe you should check also in a SDS-PAGE to see that you have protein because you don´t know if your protein is not binding to the resin or if the resin is not working with any protein.
Are you sure that you are using the correct pH regarding the pI of your protein in order to assure that it is binding to the resin?

[mirc]

>which buffer and pH do you use?

2mM Tris pH8
1mM MgCl2
50-1000mM NaCl

Unfortunately we don´t have a hydrophobic interaction column but maybe I should start off with a gel filtration, though that would be an uncommon first step...

It´s U2OS cell lysate, not e. coli, but at ph8 there should be a lot of negatively charged protein. Calculated pI of the protein is ~5, so should be negative. The dynamic binding capacity is stated as >130mg BSA/ ml bed volume, so I shouldn´t have overloaded the column, or do I misunderstood something? Also the volume shouldn´t matter with IEX....I´m using a polyclonal AB against the protein btw....

Edited by mirc, 27 May 2010 - 08:15 AM.

[Inmost sun]

mirc, on May 27 2010, 05:05 PM, said:

>which buffer and pH do you use?

2mM Tris pH8
1mM MgCl2
50-1000mM NaCl

Unfortunately we don´t have a hydrophobic interaction column but maybe I should start off with a gel filtration, though that would be an uncommon first step...

It´s U2OS cell lysate, not e. coli, but at ph8 there should be a lot of negatively charged protein. Calculated pI of the protein is ~5, so should be negative. The dynamic binding capacity is stated as >130mg BSA/ ml bed volume, so I shouldn´t have overloaded the column, or do I misunderstood something? Also the volume shouldn´t matter with IEX....I´m using a polyclonal AB against the protein btw....

you can combine cationic and anionic exchanger chromatography;  I routinely started with cationic exchanger chromatography, then hydrophobic interaction chromatography then Q column...

[paramyosin]

The dynamic binding capacity is obtained after several hours of interaction between the resin and a substrate, the real binding capacity depends on the residence time. Definitely I would try with less initial volumen, i think that you are using too much for your 1 ml column. The sample load is an important parameter in resolution. Are you using FPLC? with very high loads, sample displacement can occur. I agree about cation and anion exchange, sometimes you can have surprises regarding the real pI of your target protein.

[mdfenko]

you might want to consider increasing the concentration of tris to at least 20mM. 2mM won't do a very good job of maintaining the pH.

[Prep!]

conductivity and pH are the two factors which will decide the binding on Q column.. i don know abt bacterial products but with cell culture based products Q is a very very common first step!!!!

[mirc]

@paramyosin:
Sorry, that was typo; I do use 20mM Tris and an FPLC System
What do you mean by "sample displacement can occur"?
I will try with a 500µl loop though. I just thought that sample volume wouldn´t matter with IEX.

[paramyosin]

the sample displacement is a phenomenon that can be occur at very high loads due to overlap between solute zones which may reduce yield and/or purity of your target solute. The overload effects may be avoided by restricting the load..... However you say that you don´t have protein at all binded to the resin, have you?. Are you meassuring your conductivity?, i woul try with a initial one under 10 mS/cm and afterward you can eliminate the protein in which you are not interested in (maybe 50 mM is too much and you can loose your protein), try just to be sure that you are not loosing your target protein. Then you say that you perform a 15 CV gradiente between 50 and 1 M NaCl. I haven´t got a clear idea about your chromatogram, have you got peaks in it?, i am talking about other proteins´peaks in order to know if your column is properly working. Can you send the file of the chromatogram?

[mirc]

sorry for responding so late,
The column does bind some protein (only a little in comparison with the flow through peak (~aprox1%). If I scout for another protein via WB it seems nicely bound, but my protein of interest isn´t. The problem is, that it was retained once (my initial experiment) but ever since my protein of interest doesn´t bind any more...
Unfortunately I can´t measure conductivity...

[paramyosin]

Different proteins bind differently in a resin!, but if you have good binding of other protein is because the resin is ok. Maybe your initial conditions were different, more NaCl concentration?, different pH?, different resin-protein relation?

[NHI]

hi fren...

by the way u have to see the principle of Ion exchange chromatography...let me explain..

since u use Q resin....i do believe it is positively change resin.if u want to make ur protein bind to the resin, u should make ur protein as -vely charged.HOW?????

Do u know the isoelectric point of your protein????if not, u should make an isoelectric focussing (IEF) experiment...then, from the pI value that u will get, u can know what is the appropriate pH for ur sample to ensure the it will bind...

i give u example, let say the sample is IgG....u spin down ur sample and run IEF in order to get the pI value...
let say the pI is between 7.6 to 8.2....by assuming an electrophoretic titration curve, i.e. lower the value from 7.6 will make the protein as +vely charge, whereas increase the value from 8.2 will make the protein as -vely charge...

let say u want the IgG bind to Q resin, so u must choose pH above 8.2...let say u set pH at 8.6

Then, u choose any buffer that has buffering region at this chosen pH....at pH 8.6, Buffer Tris-HCl (10 mM) can help...then, u dialyze/ buffer exchange ur sample to this buffer...

Secondly, by rule of thumb....for ion exchange chromatography binding mode (i.e. product of interest will bind to resin), the conductivity of sample must less than 5 mS/cm...so, use buffer at low strength...such as 10mM or 20 mM....


by the way, good luck...