Protein Sample Preparation: Shoot for > 20 ODml
Posted by Peter Nollert on Thu, Oct 29, 2009 @ 02:00 PM
I learned this simple trick from Larry Miercke @ UCSF: rather than measuring OD280 and computing mg/ml using an extinction coefficient, go straight with ODs.
Here's how this works:
Measure the OD280 (that's the Optical Density at 280 nm; using a proper reference buffer) of the protein solution at hand. For simplicity reason let's say you've got 50 ml of pooled eluate fractions and you're using a 1 cm path length cuvette. If your OD280 reading is 0.5 you'd multiply 0.5 OD by 50 ml and say that you've got
"25 ODml worth of protein".
Note that as a rule of thumb for most proteins:
1. you can assume that an 2 ODml correspond to 1 mg, and an OD of 2 corresponds to ca. 1 mg/ml;
2. concentrate the protein solution to higher than 20 OD to set it up in a protein crystallization trial.
So, when you're done with a protein prep you want to keep concentrating until you've got an OD exceeding 20. Say your solution is now concentrated to an OD of 22 in a single ml - that's 22 ODml. Let's snap freeze 400 ul and use the remaining 600 ul to set up 6 x 96 1 ul+1ul crystallization experiments (Wizard I, II, III and IV and Cryo I and II of course ;) for incubation at RT and at 4C.
What do you do if someone asks for the protein concentration? Using the rule of thumb (OD of 2 corresponds to ca. 1 mg of protein) you can do the quick math: knowing that your purification yielded a total of 22 ODml, divided by 2 equals: 11 mg of protein! And since the concentration procedure resulted in a ml of solution the protein concentration you used to set up the crystallization experiment is 22 ODml / 1 ml - that's 11 mg/ml.
Pretty simple, hm? No need to employ Beer Lambert.
At first sight there's no fundamental difference between accounting in terms of ODml or using properly calculated mg/ml. There are several advantages of the ODml system though:
1. you're dealing with a tangible parameter that's easy to assay anywhere in the purification protocol,
2. It's easy to monitor the slight decrease of the ODml during concentration, if the ODml increase there's a problem,
3. you're not obligated to use a parameter that you know is associated with some margin of error since the calculated extinction coefficient is not perfectly accurate.
I've also seen how extinction coefficients for a particular protein have changed as I progressed with my project. Other than mundane OD280 errors from Raleigh Scatter and buffer or sample contamination there are actual reasons why the extinction coefficient may change during the course of purification: This may be due to the
4. Cys redox state that you're starting to better manage at one point or
5. ligands that dissociate during purification.
Going back in your notebook you may be wondering, 'which extinction coefficient did I use to compute this protein concentration'? You're not bothered by such questions when using OD and ODml.
BTW, a similar argument may be said about the molecular weight (MW) of the target: you *think* you're working with a 46 kDa protein as calculated from sequence until you've seen the Mass Spec analysis. Since the MW is part of your Beer Lambert conversion you can avoid this systematic error.
In short: ODml are for real and are simple to use.
Nevertheless, for all of you wishing to get an estimate of the protein concentration in mg/ml using amino-acid based estimated extinction coefficients, here's how to use the ProtParam tool for this purpose:
1. Paste in your amino acid sequence into the sequence box (I'm using the sequence of chicken lysozyme)
ProtParam accepts amino acid sequences, counts Tyr, Trp, Cys, adds up the corresponding extinction coefficients and outputs this number as an overall Extinction coefficient (and other data) to the user.
2. Click the button 'Computer parameters' and a data rich report is generated, listing
• Number of amino acids
• Molecular weight
• Theoretical pI
• Amino acid composition
• Total number of negatively charged residues
• Total number of positively charged residues
• Atomic composition
• Formula
• Total number of atoms
• Extinction coefficients
• Estimated half life
• Instability index
• Aliphatic index
• Grand average of hydrophathicity (GRAVY)
That's a lot of data to digest. Let's look closely at the section 'Extinction Coefficient':
The extinction coefficients are served ready to get inserted into Beer Lambert's Law, where the protein concentration is calculated from:
OD280 x MW (in g/mol)
-----------------------------------------------
Ext. coefficient (1/Mcm) x path length (cm)
3. Using the example above (OD of 22), the concentration becomes:
22 x 16,238.6 g/mol
----------------------------- = 9.4 mg/ml
37,970 /Mcm x 1 cm
That's a suitable concentration to go into protein crystallization trials with and is pretty close to the 11 mg/ml estimate using the OD rule of thumb.
Peter
