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Search our Tips from A to Z for more information. Still having trouble finding what you want? Contact Hampton Research technical support.
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| A is for Aggregation |
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| Aggregation can be a deterrent to the crystallization of biological macromolecules including proteins, peptides, and nucleic acids. The presence of sample aggregation can be detected by either dynamic light scattering or native gel electrophoresis. Aggregation might be caused by hydrophobic patches on the surface of the sample, differently charged isoforms, differently phosphorylated isoforms, mixtures of methylated and non-methylated samples, glycosylation, as well as electrostatic interactions. Aggregation can be due to autologous aggregation where the protein is aggregating with itself or heterologous contamination where the sample is aggregating with other proteins. In the case of heterologous contamination, further purification of the sample should be seriously considered. In the case of autologous aggregation that precludes crystallization one might consider:
Using molecular biology to manipulate intra and inter molecule interactions by modifying the sample sequence (alter, add, or delete residues).
Use chemical additives to manipulate sample-sample and sample solvent interactions.
Detergents
Chaotropes (urea, guanidine hydrochloride, hydrochloric acid, etc)
Electrostatic agents
Alcohols (isopropanol, methanol, ethanol, etc)
Salts (sodium chloride, potassium chloride, sodium fluoride, etc)
Polyols (glycerol, PEG 400, etc)
Ligands, inhibitors, co-factors, and metals
Use temperature to prevent aggregation (0°C and 60°C)
Consider a fusion protein
Remove C-terminus or N-terminus
Truncate domains
Remove His-tag
In some cases aggregates can be removed by centrifugation or filtration.
In some cases the aggregates can be removed by mixing the sample with the crystallization reagent, allowing the sample to incubate for 15 minutes, centrifuging the sample/reagent mixture, removing the precipitate and setting the drop with the supernatant.
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TECHNICAL SUPPORT
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Fax: 949-425-1611
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Email: tech@hrmail.com
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Hampton Research
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Tel: 949-425-1321
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