I have expressed viral coat protein in BL21 cells. After purification of the protein, I am observing two bands, one of the size of the protein and one exact double size in SDS page. I think the possibility of protein dimerization. Can you suggest how can I validate my observation?
It could be an artifact of SDS-PAGE, but here are some common methods for studying oligomerization of proteins in solution:
Gel filtration (also called size exclusion) chromatography with a calibrated column
Analytical ultracentrifugation
Dynamic light scattering
Size exclusion chromatography with multiangle light scattering detector (SEC-MALS)
It could be an artifact of SDS-PAGE, but here are some common methods for studying oligomerization of proteins in solution:
Gel filtration (also called size exclusion) chromatography with a calibrated column
Analytical ultracentrifugation
Dynamic light scattering
Size exclusion chromatography with multiangle light scattering detector (SEC-MALS)
Dear Anish Tamang ,
As size, you mean the calibrated length of the denaturated proteins or the amount of material, so the thickness of the band?
What is the size of the viral coat protein? As far as I know, most are in between 10 and 30 kDa.
How did you perform the SDS-PAGE? How are the controls?
It is possible that there is contamination, contaminants pulled down something in your purification, some protein was cleaved or extended (i.e poly-ubiquitinated) or the sample was not entirely denaturated.
If the controls are as expected, the easiest way is to run again on PAGE in both native and denaturating (SDS) conditions. Mind that the native requires different current settings.
To corroborate only the dimerization, you can perform:
- co-immunoprecipitation (co-IP): express at least one protein of interest fused to an epitope tag and purify with the proper antibody, if they're pulled down together, they're dimerized.
- Affinity Chromatography (AC): express protein of interest fused to an affinity tag (i.e. olyhistidine-tag (His) or GST) and purify them through chromatography accordingly.
To corroborate dimerization and obtain sub-cellular localization you can use bimolecular fluorescence complementation (BiFC), expressing the proteins of interest tagged with non-fluorescent fragments of either a FRET (Förster Resonance Energy Transfer) construct (i.e. CyPet - YPet) or a self-assembling fluorescent protein (i.e. saGFP).
If the non-fluorescent fragments are carried into proximity they reconstitute the entire molecule and emit fluorescent signal detectable through confocal microscopy.
Since there are antibodies against fluorescent protein, you can combine BiFC and co-IP.
If they do indeed dimerize, I suggest a control to confirm the specificity of the interaction.
Other alternatives require dedicated equipment
- Analytical ultracentrifugation (AUC): it measures the radial concentration gradients of molecules, in the range 1 kDa-10 mDa (1 nm - 1 μm), generated by the application of centrifugal force at 40.000-60.000 rpm.
Chapter Analytical Ultracentrifugation
https://www.sciencedirect.com/topics/chemistry/analytical-ultracentrifugation
- Dynamic light scattering (DLS): it measures the translational diffusion coefficients Dt of particles in solution by quantifying dynamic fluctuations in scattered light, then sizes and their distributions are calculated in terms of hydrodynamic radius Rh or hydrodynamic diameter dh in the range 0.2 nm - 5.000 nm.
Chapter Dynamic Light Scattering
- Size Exclusion Chromatography (analytical SEC) using U(HPLC) columns: the technique pumps a pressurized liquid solvent containing the sample through a chromatography column filled with a solid adsorbent highly porous matrix. Thus, the molecules elute in decreasing order of apparent size (mass and shape/lenght).Once separated, the sample molecules’ sizes can be confirmed using 2 methods:
- Calibration curve: Various molecules of known size are injected onto the same column as the sample. The retention time of each component is obtained from the calibration chromatogram and plotted against the logarithmic molecular mass.
- Static light scattering: Molecular masses are calculated based on a light scattering signal and a refractive index signal and are independent of the hydration shell thickness of the molecules.
Poster Size Exclusion Chromatography
Article Mobile Phase Containing Arginine Provides More Reliable SEC ...
Article The Critical Role of Mobile Phase Composition in Size Exclus...
ElectroSpray Ionization - Mass Spectrometry (ESI-MS AKA electrospray mass spectrometry (ES-MS))
Protein dimerization can be validated in ESI-MS under non-denaturing conditions. Make sure to use lower source temperature and cone voltage.
You can also simply run SDS-PAGE under reducing and non-reducing conditions, dimeric band would probably disappear under reducing conditions. All the best.
The easiest way to confirm dimerization is by Native SDS-Page (under non-reducing conditions as proposed by Dr. Sreedhar.)
Best results are obtained with samples non-submitted to heat and in the absence of SDS and betamercapto, on this conditions you should see a majority amount of your dimer in comparison with the monomer.
On SDS-PAGE theoretically you should not see dimers since proteins get denatured. You might be seen something else, like a contaminant pulled down in your purification or maybe your protein modified (i.e poly-ubiquitinated), nevertheless could be possible, but less likely, that you are not heating properly your sample before loading or some problem with your loading buffer...sometimes I have seen higher bands that correspond to complexes not denatured, but usually they are not major bands.
One of the easiest thing to prove dimerization that I do using the same technique of protein expression in E.coli, is to co-express two different tagged constructs of your protein of interest (i.e. GST and His). If there is dimerization when you do separated purification (pulling by His or GST) or sequencial tandem affinity purification, you should pull down at the end both proteins (His and GST tagged) and capable to check by western or even band shift in your Coomassie stained gels. Of course you can use other methods like 2D Electrophoresis (combining Native-PAGE with SDS-PAGE), or Gel Filtration or the fancy techniques like FRET that someone suggested you, but that depends of your project goals and how farther you want to push it.
good luck!
Dimers should not be conserved if you're running an SDS-PAGE sample, as your proteins are denatured in these.
The extra band might be due to contamination in your purification (if you're doing a single step purification you are unlikely to remove all contaminants) or, if your gel is not reduced, might be that your protein is oxidized and running at a different size. Make sure your reducing buffer is not out of date and also confirm that both bands are your protein of interest by running a western blot (normally using an antibody against the affinity tag used for purification).
When it comes to confirming if it is a dimer, the easiest way, depending on the equipment available, is running a native gel and/or size exclusion chromatography, which are both suggested above. In the native gel you should see multiple bands corresponding to the different species (monomer/dimer/etc.), while size exclusion chromatography should give you different peaks corresponding to the different species (which can then be run on an SDS-PAGE gel to confirm that it is your protein).
As you are working with a viral coat protein, I would expect these to form multimers (not just dimers). Make sure you desalt your protein post purification as the salt content might inhibit multimerisation.
Best of luck!
The easiest way to confirm that you have your protein in both bands would be to run a western blot. In case the upper band really contains your protein in a dimeric form, you should get a signal in both positions on the gel (upper and lower running band). SDS should in theory denature your proteins to monomers but in practice you can sometimes observe dimers too (this I saw for natural dimers only). Include a lot of DTT to make sure all your cysteines are reduced and you don't have an artificial dimers made through coincidental s-s bonds.
#DLS could be one solution.
#The addition of some extra DTT or BME and boiling to denature/break all the disulfide bonds would be another solution.
Dear Kou,
Specific protein dimerization is integral to biological function, structure and control, and must be under substantial selection pressure to be maintained with such frequency throughout biology. To characterize the association of protein structures into homodimers using coevolutionary couplings extracted from Direct Coupling Analysis (DCA) in combination with Structure Based Models (SBM) are suggested.
Most accurate conformations of the structurally diverse dimeric complexes studied the mean and interfacial RMSDs methodology is also able to identify and validate with resolution, distinct dimerization conformations as for the case of the family of response regulators, which dimerize upon activation. The identification of dimeric complexes can provide interesting molecular insights in the construction of large oligomeric complexes and be useful in the study of aggregation related diseases like Alzheimer’s or Parkinson’s. In this method , Sequence Alignments and Directly Coupled Residue Pairs, Structural Modeling and Molecular Simulations are quite popular for Prediction of multiple dimeric complexes in response regulator proteins.Ides can be found with to study residue-residue coevolution involved in dimerization with structural data using equipment such as X-ray crystallography or NMR).
Ashish
Dear Anish,
Specific protein dimerization is integral to biological function, structure and control, and must be under substantial selection pressure to be maintained with such frequency throughout biology. To characterize the association of protein structures into homodimers using coevolutionary couplings extracted from Direct Coupling Analysis (DCA) in combination with Structure Based Models (SBM) are suggested. Most accurate conformations of the structurally diverse dimeric complexes studied the mean and interfacial RMSDs methodology is also able to identify and validate with resolution, distinct dimerization conformations as for the case of the family of response regulators, which dimerize upon activation. The identification of dimeric complexes can provide interesting molecular insights in the construction of large oligomeric complexes and be useful in the study of aggregation related diseases like Alzheimer’s or Parkinson’s. In this method , Sequence Alignments and Directly Coupled Residue Pairs, Structural Modeling and Molecular Simulations are quite popular for Prediction of multiple dimeric complexes in response regulator proteins.Ides can be found with to study residue-residue coevolution involved in dimerization with structural data using equipment such as X-ray crystallography or NMR).
Ashish
Anish, this sometimes is a "kinetic" artifact of reducing conditions. Not all proteins that have disulfide bonds to dimerize them are IMMEDIATELY reduced to their monomeric states, especially if the disulfide bond is protected from contact with your reducing agent. It takes time. For many proteins, that is a vanishingly small amount of time, and you get reduction to monomers on what is an instantaneous time frame, when you compare it to how long it takes to load the well in a polyacrylamide get. But that isn't the case for all proteins. You can always reduce the amount of time by warming your samples, but this sometimes causes other damage that you want to avoid. You can increase the efficiency of reduction by waiting longer (a few more minutes usually suffices) or by increasing the concentration of the reducing agent. In fact, if the dimer is protected, and money is not an impediment to you, you can do a range of reducing agent concentrations to observe the amount of reduction you get, because you will see an inverse relationship between the amount of reducing agent and the amount of dimer/monomer ratio that is seen in the gel. It's not quantitative, but it is still revealing.
Anish Tamang
You can validate your protein diamerization by optimizing its analysis by anyone of the techniques mentioned below:
Native PAGE
Analytical Ultracentrifugation ( AUC)
Dynamic Light Scattering ( DLS)
NMR
Gel Filtration etc.
All the best
Dear all, thank you for your response. But can I validate dimerization through western blotting with an antibody specific to the protein?
You cannot VALIDATE dimerization by Western blotting, unless you have two different monoclonal antibodies with two different reporters, one of which ONLY binds to a dimer, and one of which ONLY binds to a monomer. Even so, it's a terrible method for quantification. Analytical ultracentrifugation would be ideal for what you describe, if you have the instrumentation. Do you have anything besides antibodies and gels to work with? (Note: Western blotting can provide circumstantial evidence of dimerization, but still will never quantify it. Just thought I should give full disclosure in my answer.)
An approach that is compatible with Western blotting is chemical crosslinking. The idea is that if the protein is dimeric, it is easy to generate covalent crosslinks using chemical crosslinking agents, such as BS3, even at low protein and crosslinker concentrations. This is a bit subjective, because there is no specific protein or crosslinker concentration that is the cutoff for demonstrating dimerization. Therefore, crosslinking can support evidence from another method, but should not stand alone. Nevertheless, for an example of this approach, see this paper:
Article Dimerization of isolated Pseudomonas aeruginosa lipopolysacc...
Hi Anish,
sometimes in SDS-PAGE, if The boiling of sample Is not complete then Oligomer formation is possible. to confirm the dimerisation or oligomerisation you can run Native page. You can also check on analytical SEC or SEC-MALS. Western blot would help you to check for the contamination Band or specific band of the expressed protein. However, it is worth running native page for the western blot.
all the best
Blue Native PAGE works quickly to show if you have a dimer, but you'll need markers of known size. Cross-linking works too with Mass spec e.g. cross-link with DSSO, but you'll also need a positive control (something like NADOX). Finally a general size exclusion should do the trick (but I would still confirm with another method that this is your protein appearing as a dimer).
It's also worth noting that generally you don't see dimers on SDS PAGE and that what you're seeing could be a chaperone protein...
Dear Anish,
is you protein contain hydrophobic regions (eg transmembranes) or many cisteines?
In general, soluble proteins run as monomer in reducing SDS-page but of course is it possible to have some exception.
In case that you protein contains cysteines and you think that the dimerizaion is S-S mediate you can try to load also a sample with-out reducing agent to see if the ration dimer-monomer change and you the intensity of the monomer decrease.
To prove that the both bands are the same protein, you can have some different approaches:
1) Is you protein contain a tag? eg His tag? because in this case you can try to perform a WB with a antibody able to bind the tag.
2) you can try to do the WB with a policlonal antibody specific for the protein, if it is avaialble commerically
3) you can send the bands cutted from the gel to mass spectrometry analysis (gel extraction- trypsinization and Mass spectrometry peptide analisys - eg https://www.alphalyse.com/protein-identification/ ). in this way you can find out the theoretical sequence of both bands and undertand it the high MW is the dimer or one impurity. Of course this is probably the most expensive approach if you do not have a mass spectrometry team able to perform this analisys for you in your institute and you have to outsource it.
good luck
Manuele
Hi Anish,
The presence of dimers can be determined by the peak of gel filtration.
An easy method would be dynamic light scattering, which can be superior to SEC which can fail to observe some transient interactions. However, the most sure foolproof way (if expensive) is to have the bands cut out and analyzed by MS-peptide fragmentation.
https://www.cell.com/trends/biochemical-sciences/comments/S0968-0004(04)00234-8
Another strategy is to add two different affinity tags(e.g FLAG and HA) to your protein and then perform co-immunoprecipitation. If FLAG-Protein immunoprecipitate HA-Protein then they are most likely to form dimer.
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