antibody purification - ammonium sulfate precipitation - (Jun/28/2010 )

I found the following ammonium sulfate precipitation protocol online:

1. Harvest the cultures 6 days after transfection.
2. Spin down the supernatant to get rid of cell debris at 3000 rpm for 20 min.
3. Add ammonium sulfate in the amount equal (in weight) to 60% of culture. (Ex: if your culture final volume is 1 liter, add 600 g). Ammonium sulfate is added gradually, divided in 4 parts (1 part per hour) while the supernatant is being stirred slowly at 4 C. The supernatant should be mixed O/N slowly.
4. Spin down the supernatant at 4000 rpm for 30 min. and resuspend the pellets in cold PBS. The slur must be re-spun or lots of antibody will be lost. For 1-1.5 L of original (confluent) culture, the final volume in which pellets should be re-suspended should be approx. 50 ml.
5. Spin again for 10 min. at 3000 rpm to remove all particles.
6. Add 1 tablet of protease inhibitors (Complete, EDTA-free, cat # 1 873 580, Roche) dissolved in 1 ml of PBS to 25-50 ml of the suspension.
7. Add the volume of the tube to a dialysis membrane and dialyze against 2 L of cold PBS for 2 hours. Change the dialysis once more and dialyze for 2 more hours.
8. Remove the volume from the dialysis membrane and add 1 more tablet of protease inhibitors (dissolved in PBS).

I am interested in following this protocol for my first step in my antibody purification (The second step would be a protein A column). I was going to make the following change: in step 4 I was going to use 20mM (or maybe 50mM) Tris pH 7.4 to resuspend the precipitate. The reason is that this buffer will be low salt (compared to PBS) and would I was thinking it would resuspend the pellet easier.

One question: what is the reason for step 5? Isn't is possible that some antibody is still precipitated after resuspending the pellet? Shouldn't you dialyze the excess ammonium sulfate first? Or is that precipitate only other proteins besides the antibody?

Just out of curiosity... do u even need to do this before loading onto the protein A column!!!
Cant u directly load??!!!!

Prep! on Jun 29 2010, 03:05 AM said:

Yes, I absolutely can. The recommended column protocol says to dilute your supernatant 1:1 with PBS and then run through column at 1ml / min.

So, I am producing 4L of supernatant. After adding PBS it will be 8L; at 1ml / min it will equal 8000min = 133hrs = 5days.

Instead I wanted concentrate the 4L into 200ml in PBS; then I can run it in about 3hrs!!!!

Also, if I remember correctly, antibody binds better to the protein A column at higher concentrations (so maybe better yields?).

Well, that is my reasoning.

Any suggestions are welcome.

well i guess u are using the wrong dimentions of column then.. cause a proper dimention shud let u load a higher flow rate quite easily... are u planning to load all the harvest on a small column (say a high trap or sumthing)... just take care its not ablove the binding capacity of the column... it wud be better if u can provide more details.. i think tere is sum error here...

Disclaimer: I m not a process person but have worked closely with many!!

Prep! on Jun 30 2010, 04:39 AM said:

I am going to use pierce protein A cartridge, 1ml column. According to specs it can bind 35mg of human IgG. Here is the link for the instructions:
protein A cartridge instructions

I was planning on connecting the column to the FPLC machine and following the "Procedure for Antibody Purification using a Liquid Chromatography System" section of the instructions. Step 3 says to use 1ml/min for the 1ml column. It does say I can use higher speed for the 5ml column. So in that respect you are right. The problem is that I am not the one that gets to approve orders in the lab. The usual response from my boss is: "find a cheaper way of doing it". Usually when I have done it in the past, I am able to obtain about 10 - 12mg antibody from 2L culture supernatant. Thus I am expecting to get around 24mg this time around - below the 35mg of the 1ml column. Also, for $160 I get two columns; for $320 the 5ml column will cost 4X more.

Well that is my reasoning for things.

Like I stated earlier, I am open to suggestions.

i thought so!!!
well then i guess your stategy is fine (as i said i cant help u more on the precipitation part!!!)... i just hope u dont lose ur antibody during precipitation!!!

also try to convince ur superiiors tat along with cost time also matters!!! in fact it can compensate the extra bit of money tat u put in!!!

Best luck!!

Prep! on Jun 30 2010, 09:17 PM said:

I once tried the saving time = saving money argument. It just doesn't work in research. Why? Simply put, if you work extra hours you don't get paid extra.
And it is not only my boss, many PIs are the same way. I once had a friend that was trying to convince his PI to buy pre-cast gels because it would save time and hence save money. The PI told him: "no because saving time doesn't save money - work 40hrs or work 80hrs per week you get paid the same amount of money".

I guess it was bold and rude statement, but nevertheless its the truth.

Sometimes I wish I had studied something else...

Anyways, thanks for your input. I guess the "smart" thing to have done would be to pack a 20ml column by myself. I have only packed a column once before though.

hey tat s fine chicho... dont start hating wat u doing!!! may be u are best doing wat u are right now and so u not doing anything else!!!
these might just be the learnings tat u are gonna take once u thru with ur course!!!
Best luck!!!
Do come back with wat results u got!!!