I have been trying to label one protein which I purified recently. And I am facing a lot of troubles. I will describing my process in details. And I will be highly obliged to receive any suggestion.
My protein is stable as a dimer and the monomer mass is 19.2 kDa. I want to label it with a Cy5-malemide dye at a Cys residue. However, the rick is I want one Cy5 label per dimer.
I am using TRIS buffer, maintaining the pH around 7.2. As a reducing agent I have TCEP in the buffer (almost 50-100x compared to the dimer concentration). I do take care in maintaining degassed environment.
I have tried different protein(dimer):dye ratio from 1:3 to 1:6.
I do the reaction for 2 hours in room temperature and then overnight at 4C.
But always I get a labeling efficiency of 50%. As you can understand that is way below what I desire and I really need suggestions how to improve.
Hi Kushal,
Although I probably cannot provide a direct solution to your issues, I have a few questions:
First of all, how do you determine your labeling efficiency? And isn't 50% what you're after (1 label per 2 protein molecules)? Or does 50% mean that half of your dimers contains one label?
Is the cysteine you're targeting involved in a disulfide bond? Or is it capped? And is it solvent-exposed? You're not labeling under denaturing conditions, correct?
Why are you using these reaction conditions? Have you tried shorter incubations at a higher ratio (and perhaps at a higher temperature)? I'm trying to compare your conditions to those routinely used for cysteine alkylation.
It is known that maleimides can hydrolyze in aqueous media at pH 7-8 making them non-reactive to cysteines.
Hope this helps (a bit)!
Hi Eef,
Thank you for going through my problem. I will try to reply to your questions.
The labeling efficiency I am saying is wrt to dimers. And I must add it is hard to completely separate the unlabeled from the labeled through FPLC columns. And thus I need to increase my labeling amount.
The Cys are very prone to form disufide bonds, but our natuve protein does not have that. In case to prevent them we add TCEP. I did check long back about the solevent exposure. It's not completely solvent exposed though.
I am looking really for suggections on the conditions. I tried this condition as one group previously tried to label some other protein similarly and did this conditions. So I thought of take that as reference.
I read the protocols from the companies for this labeling with Cy5-malemide and they suggest pH7-7.5.
I hope my answers help you to take a more deeper look.
If you are planning to use Cy5 as the donor in a FRET experiment and measure the donor fluorescence, then it is not really a problem if the labeling stoichiometry is lower than the ideal value, because unlabeled protein is invisible in the experiment. It is a bigger problem if the Cy5 is to be used as the acceptor.
Increasing the label to protein ratio should increase the labeling stoichiometry, but at the risk of increasing the amount of dimers with 2 labels attached.
You should surely remove (by ultrafiltration or sephadex micro-column) or quench (by adding 4-azidobenzoic acid) the TCEP reagent, because it reacts with your maleimide and so quench your reagent! I will not suggest reaction at higher temperature, normally 2h at 4 C is enough, otherwise you will have a lot of unspecific labeling through Lys residues
Dear Adam, for my experiments Cy5 is the acceptor.
Dear Igor,
You mean remove TCEP immediately after the reaction right? After the labeling reaction I run the whole sample through a superdex column and thus I think it is removed.
No, I mean to remove before an addition of Cy5-maleimide. React your protein with TCEP (10eq) for 1.5h at 37°C, then remove using Vivaspin column 10kDa or Biospin column 6 kDa. Then check concentration and add maleimide 10 eq. for 2h at 4°C, then purification
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