DEAE has a pKa 6.9 (DE52)
Tryptophan synthase, alpha subunit has pI 5.31
Wash buffer & Equlibriation buffer are same pH 7.8 (10mM phosphate buffer 4mM Na2EDTA 1mM DTE).
Pellet fraction (inclusion bodies) is resuspended in (100mM, 1mM Na2EDTA, 1mM DTE) wash buffer and after centrifugation 45min twice into urea buffer pH 7.8 (6M urea + wash buffer).
Both supernatant and pellet fractions are dialysed for 4 hours.
50mM phosphate buffer, 2mM Na2EDTA, 1mM DTE and 25mM 2mM Na2EDTA, 1mM DTE for 4hrs, Finally with wash buffer (10mM phosphate buffer, 2mM Na2EDTA, 1mM DTE).
When we load these sample on to the column protein (pI 5.31) is negatively charged in pH 7.8, DEAE (pKa 6.9) is also negatively charged in pH 7.8 how does binding happen and it is eluted with increased concentration of EDTA 4mM, 10mM phosphate buffer, 1mM DTE. Even nucleotides don't bind to DEAE?
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For purification of reverse transcriptase enzyme.
After affinity purification using HisTrap HP 5ml we run pool fractions on SDS
then on Sephadex G25 column (equilibrated with storage buffer pH 8)
then after SDS, we do IEX DEAE-Sepharose CL6B (equilibrated with storage buffer pH 8 and washed/eluted with the same buffer).
The purpose of using IEX is to remove nucleotide contaminants.
RT pI is 8.6 DEAE, pI is 6.9 DEAE equilibrated in buffer pH 8 will make it negatively charged, meanwhile RT in buffer pH 8 will be positive in charge thus binds to DEAE?
How are nucleotide removed and we don't use any elution buffer?
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Finally, what does Sephacryl S100 mean in practice, for example, a protein taq enzyme 95kDA after AS 60% saturation is passed through it will pass through the pore or void volume?
1. I do not think deae has a pka value of 6.9. It must be greater, thus at pH 7.8 the adsorbent is positively charged.
2. Do not use phosphate buffer as it will compete for the binding sites.
3. A 95 kDa protein will not enter the pores of sephacryl s-100 hr
XK is just the identification code given by pharmacia, you are right about diameter and height.
The protein should not be close to 100,000 if you want it to enter the pores.
okay, but the range is 1000 to 100,000 for proteins close to max value find it hard to pass through the pore and for proteins ranging from 1000, 30,000, 50,000 daltons all enter the pore? why is the range from 1000? anything less than 1000 can also enter the pore. what exactly does 1000 indicate.
and gel filtration is always about eluting protein through void volume or is there more about it?
Proteins in the range between 1000 and 100,000 will (in theory) enter the pores and be separated by size. Below 1000 all molecules have access to all pores thus no separation occurs, above 100,000 all molecules are forbidden from the pores thus no separation occurs.
Gel filtration is to recognize what I just wrote. How you perform the separation is up to your mixture.
So in principal 1000 dalton protein reaches bottom quickly that 60,000 dalton protein. But in practice 95000 dalton protein finds it hard to pass through the pore and struggles its way to make it to the bottom by avoiding the pore (void volume) or passing through the pore meanwhile salt molecules anything less than 1000 reach the bottom and gets eliminated therefore it is a good way to polish protein from anything that is way smaller than 95000 daltons.
I think you have it reversed:
Larger proteins (greater than 100Kd will emerge from the column first in the void volume. The void volume is generally about 42% of the total column volume. Any protein smaller than 100Kd will spend some fraction of the time within the inclusion volume so those molecules are retained in a larger volume and will elute after the void volume. Therefore, Larger molecules (greater than pore) elute first - Retention time is inversely proportional to molecule size.
I usually measure the total column volume by the salt peak (those Molecules are much smaller than the pore of the bead) so you will get a slight bump in conductivity at the end of the run.. Therefore, I design my sample to have a little higher salt concentration than the running/equilibration buffer of the column. I measure retention time by both time and flow rate * time is a measure of volume. The ration of elution volume/total volume will allow normalization between chromatographic runs - this will facilitate the optimization of your flow rates for higher resolution between peaks.
Look forward to hearing back about your results
Best regards
Dan
We use S200 not S100
S200 range ~5 × 10^3– ~2.5 × 10^5 (5000Da-250kDa). I noticed all sephacryl 100 to 500 have same particle size (50 micro meter) how do they differentiate different protein sizes that pass through it. I hear about the degree of swelling although it's not clear how that works. and S200 exclusion limit give approx 30 for DNA
Greater than 95 kDa? this is iptg expressed protein and AS precipitate 60% followed by IEX then again 60% AS precipitation then passed on to S200
All this time I was thinking there wouldn't be much proteins greater than 95kDa after second AS precipitation (we don't run a gel at this step)
This is how my procedure is after second AS ppt, equilibration S200 column buffer contains (hepes buffer 20mM, KCL 50mM, EDTA...etc) load 2ml sample flow rate 1.3 ml/min elute with same buffer same flow rate and collect the peak.
Chromatographic separations depend on the size of the adsorbent, the larger the size the poorer the separation. For gel filtration the separation occurs within the adsorbent. The size of the adsorbent only affects the diffusion length. Smaller particles will produce narrower peaks but will also increase the required pressure.
The working range for sephacryl is given for the swollen gel. If you use acetone as solvent, for instance, you will have a different working range.
If you are loading 2 mL of sample I assume you have a 100 mL column, is this so?
it is a 300ml column
"For gel filtration, the separation occurs within the adsorbent." right but all have the same pore size? diffusion length meaning it would minimize sample diffusing horizontally?
So swollen gel is key for separation, the degree of swelling facilitates separation based on MW how do we determine that anyway?
No, different size exclusion media have different distribution of pore sizes.
Using larger particles implies that the diffusion length within the pores is larger, thus it takes more time for the molecules to enter (if they can) and leave the pores; which ends up in broader peaks.
Do not forget that you are using a gel, thus the presence of water is part of your separation system. The degree of swelling is fixed for a particular solvent according to the preparation of the polymer that constitutes the adsorbent. You just buy the product that is supposed to work for your needs.
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