This is difficult to answer, but if you can provide material that is 95% or higher purity as estimated by assays such as gel electrophoresis, mass spec. analysis or other analytical chromatography technique, this will give the best chance of success.
If you are providing us with your macromolecule, use a buffer, which contains the minimal number of components (buffers, stabilizing agents, salts) and lowest concentrations of those components that stabilize your molecule. Every marcomolecule is different and therefore may require different buffers. 10-20 mM TRIS/HEPES/MES etc. buffering agent with 50-100 mM NaCl is a good place to start. Your sample may require additional component such as Mg2+ for oligonucleotide samples, glycerol or ethylene glycol for samples that aggregate easily, reducing agents such as DTT or TCEP etc. Small molecule compounds should be provided as a lyophilized powder when possible and as a high concentration liquid when necessary. If DMSO or DMF are in your small molecule solution, we need to know how much.
Typical/Example buffers by assay:
- Crystallography- 10 mM Tris pH 8.0, 25-100 mM NaCl
- SPR- 50 mM Hepes pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% Tween/ or PBST (avoid components with high refractive indices and high viscocities when possible such as DMSO, sucrose, glycerol etc.)
- CD spectroscopy- low concentrations of UV-Vis inactive buffering agents. Avoid chloride, citrates, MOPS, immidazole and DTT as these components absorb in the visible and/or UV region. Phosphate and Tris buffers are common (do not pH with HCl). De-gas all buffers prior to analysis.
- AUC- PBS is commonly used. Ensure that all biomacromolecules are dialyzed into the same batch of buffer. Provide the Core staff with a small volume of your dialysis buffer for dilutions and reference channels.
- ITC –PBS or Tris are commonly used depending on the type of interaction that you are studying (proton dependent or independent). Dialyze all samples into the same batch of buffer and provide a sample of that buffer to the core staff along with your sample. Degas all buffers and samples immediately prior to analysis.
- KinExA- PBS is typically used as a buffer. BSA will be added to samples prior to analysis.
- DSF- flexible buffers, but HEPES is typical.
This question will require a consultation with a Core staff member to determine. The answer depends on your assay and your biomacromolecule. When you meet with a core staff member to discuss the experimental design, be sure to mention if you have a reagent which is a limiting factor. Perhaps the assay can be designed to minimize the usage of your more precious reagent. Very generally speaking, 10-20 mg for crystallography, 1-10 mg for ITS/DSF/CD/KinExA and 100 ug-1mg for SPR.
Please label your tubes legibly and include important information such as a) name of sample, b) concentration of sample and c) buffer components and concentrations. It is also helpful if you can provide us with as much information as you have with your macromolecule such as any know information about purity, how stable it is in different pH salt or temperature conditions, how high it can tolerate being concentrated etc. The more information you can provide, the better. Also let us know how we should store the protein prior to and during experimentation.
If you are delivering your protein to us personally, please bring it on ice if it is in a thawed state already or bring it frozen on dry ice if it was previously frozen or a lyophilized powder. If you are shipping us your material, email us in advance to ensure that we have someone here to receive it. Ship it overnight either on ice or frozen on dry ice.
For most of our instrumentation, the answer is yes. If you are interested in running your own samples and learning these techniques, please contact a core staff member and arrange for a time to meet and assess the amount of training that you will likely require based on previous experience. Once training and familiarity is established you will be an approved user, with access to the instrumentation at your convenience. However, if you are not interested or don’t have the time to put into training on each instrument, leave the work to us. We are happy to characterize your samples on your behalf.
For instrumentation that requires gas (X-ray diffraction and CD spectroscopy), please contact a core staff member at least 2 days in advance so that we have time to order new gas tanks if necessary. For all other instrumentation review the instruments availability using our iLab reservations tab and contact a core staff member to reserve time on your behalf.
The core provides you access to pipets, tips, tubes etc. along with most of the items you would need for a typical experiment on an instrument (examples include plates for crystallography experiments, quartz cuvettes for CD, tubes for SPR etc.). If you have an uncommon protocol or special needs/reagents, please plan to bring those items with you or contact a core staff member to ask if we have those items in stock.