Thioflavin T assay could be used for detecting amyloid fibrils. This is useful only if your protein forms fibrils. Else Size Exclusion HPLC could be helpful.

Hi there,

An easy assay is to monitor protein aggregation in time by following light scattering at 500nm in a fluorometer (as in the citrate synthase aggregation assay used for chaperone activity  assay). µM concentration is required. 

As Dominique  stated light scatter (at a variety of wavelengths usually >320nm) is often used to assess aggregation.  Its sensitive enough if you have a concentrated solution of your protein. More sophisticated techniques such as DLS can give insight into actual aggregate size etc. at much lower concentrations. Low speed centrifugation behavior and passage through a MWCO spin filter are also convenient ways to asses aggregation. Simply monitoring the A280nm signal as the protein of interest is moved through a 2X larger MWCO filter can give be very useful to screen for buffer conditions which disfavor aggregation.

Rayleigh light scattering in the ultraviolet absorbance spectrum is a good indication of aggregation. Most proteins have a peak at about 280 nm that cuts off above about 320 nm. If you see a broad peak that doesn't cut off, it is a sign of aggregation. It's basically a UV turbidity measurement. If you can see turbidity by eye (in the visible spectrum), the sample is highly aggregated.

Large aggregates can be pelleted in a centrifuge faster than the unaggregated protein, so comparing the UV absorbances of centrifuged and uncentrifuged samples can indicate whether any aggregates are present.

If you have a dynamic light scattering instrument available, it is a very quick and sensitive way to test for aggregation, requiring about 1 minute and a very small volume of sample that can be recovered from the cuvette. However, it can be too sensitive, because it is much more sensitive to large particles than to small ones, so that even a few percent of aggregates overwhelms the signal from monomers. Also, any particulate contaminants in the sample will mess up the measurement. The sample should be centrifuged to remove dust and other large particles. Of course, this may also reduce the amount of the aggregates you are trying to detect.

Another method is size exclusion chromatography (SEC), especially when combined with a multiangle light scattering (MALS) detector. With SEC alone, and molecular weight standards, the molecular weight of the protein can be measured based on its elution volume. The shape of the protein can also have an influence, however. With the MALS detector, the shape of the protein doesn't matter, and no standards are required. You can read out the molecular weight of the protein as it elutes from the column. Since it requires no standards, isn't affected by shape, and can be done under physiological conditions, SEC-MALS is a superb method of analyzing your protein sample for aggregation. In combination with a UV detector, it can give quantitative information about aggregation, too.

Aggregation and self-assembly of proteins is typically concentration dependent, so if your protein contains a tryptophan (or several) you can typically just see if you have a time and concentration dependent loss/gain in fluorescence (depending on the nature of the aggregate). 

try a SDS-Page or a native gel to see if you have any type of aggregation (it may be just precipitation of unstructured aggregates), and if you see larger bands than your monomer, then do ThT to see if you have fibrils (oligomers/oligomeric species have lower ThT affinity or no affinity at all). For time course motorization of aggregation you can use SLS (static light cattering) or DLS (dynamic light scattering) but be aware that for DLS you would a very very clean sample to start with (only a monomeric peak should be in the beginning)

Hi Peter,

The simplest way detect the protein aggregation would be to dissolve the protein and incubate under the given conditions and then centrifuge the solution at nearly 100000 g. The check the protein concentration (using Bradford, Lowry assay or Absorbance at 280 nm) in the supernatant. If the concentration in the supernatant is less than the protein concentration in original solution, it is an indication that some of the protein has undergone aggregation . Using this method you will be able estimate the extent of the protein aggregation.

Congo red may be an ideal dye to detect the aggregated protein. Rather, it is generally used for detection of amyloidogenic nature of protein aggregates. The protein aggregates are amyloidogenic in nature you will observe a red shift in the absorption maximum of Congo red. But you are not likely to observe any shift if the aggregates are non-amyloidogenic.

In addition, you can use all the analytical techniques that are mentioned above by different expert to assess the protein aggregates, quantitatively.

Best wishes

Hi Peter,

Based on my experience, first I suggest you try light scattering if your aggregation is homogeneous. Second, analytical ultracentrifugation is better if your protein oligomerization is large than ~200kDa. Actually, you can also try the turbidity assays, but wavelength at 405nm, it is more sensitive than OD600.

I hope these will help!

hello Peter Rehbein,

you can use several biophysical tools to determined the protein aggregation and aggregates morphology. i am pasting few reference below kindly read it. it would be helpful for you.

Cetyltrimethylammonium bromide (CTAB) promote amyloid fibril formation in carbohydrate binding protein (concanavalin A) at physiological pH. JM Khan, MS Khan, MS Ali, NA Al-Shabib, RH Khan.RSC Advances 6 (44), 38100-38111.

JM Khan, P Sharma, K Arora, N Kishor, P Kaila, P Guptasarma. The Achilles’ Heel of “Ultrastable” Hyperthermophile Proteins: Submillimolar Concentrations of SDS Stimulate Rapid Conformational Change, Aggregation, and Amyloid Formation in Proteins Carrying Overall Positive Charge. Biochemistry 55 (28), 3920-3936