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CO-310 | 1 Kit | 302,10 | Add to Basket/Quote Add to Notepad |
For general laboratory use.
Shipping: shipped at ambient temperature
Storage Conditions: store at 4 °C
Shelf Life: 12 months
Applications:
The JBS Solubility Kit is a pre-crystallization screen to improve the composition of the initial protein buffer solution prior to performing crystallization set-ups [1].
Description:
Since the highly complex properties of proteins are dependent on their environment, buffer solutions play an important role, i.e. influencing the solubility and the aggregation behaviour of the protein sample. Studies have shown that aggregation of the protein may inhibit nucleation and crystal growth.
Therefore, the JBS Solubility Kit has been designed to investigate protein samples towards their homogeneity and monodispersity in dependence of the buffer solution, employing hanging-drop vapour diffusion experiments combined with dynamic light scattering.
Composition:
The JBS Solubility Kit comprises two individual kits for successive use:
A: Buffer Kit
24 buffer solutions at different pH values, 100 mM, supplied in 10 ml volumes
No. | Buffer | pH |
1 | Glycine | 3.0 |
2 | Sodium Citrate | 3.2 |
3 | PIPPS | 3.7 |
4 | Sodium Citrate | 4.0 |
5 | Sodium Acetate | 4.5 |
6 | Sodium / Potassium Phosphate | 5.0 |
7 | Sodium Citrate | 5.5 |
8 | Sodium / Potassium Phosphate | 6.0 |
9 | Bis-Tris | 6.0 |
10 | MES | 6.2 |
11 | ADA | 6.5 |
12 | Bis-Tris Propane | 6.5 |
13 | Ammonium Acetate | 7.0 |
14 | MOPS | 7.0 |
15 | Sodium / Potassium Phosphate | 7.0 |
16 | HEPES | 7.5 |
17 | Tris | 7.5 |
18 | EPPS | 8.0 |
19 | Imidazole | 8.0 |
20 | Bicine | 8.5 |
21 | Tris | 8.5 |
22 | CHES | 9.0 |
23 | CHES | 9.5 |
24 | CAPS | 10.0 |
B: Additive Kit
14 Additives solutions, supplied in 250 μl volumes, ready to use, concentration adjusted
No. | Additive | Concentration of Stock Solution |
1 | Sodium Chloride | 80 mM |
2 | Sodium Chloride | 200 mM |
3 | Sodium Chloride | 400 mM |
4 | Glycerol | 20% |
5 | Glycerol | 40% |
6 | CHAPS | 8 mM |
7 | Octyl Glucopyranoside | 0.4 % |
8 | Octyl Glucopyranoside | 4% |
9 | Dodecyl Maltoside | 0.4 % |
10 | Dodecyl Maltoside | 4% |
11 | BME | 40 mM |
12 | DTT | 4 mM |
13 | DTT | 20 mM |
14 | TCEP | 120 mM |
Instructions
The screening for a suitable buffer solution is performed in three successive steps:
(1) Hanging-drop experiment
(2) Dynamic light-scattering analysis (DLS)
(3) Additive Screen
(1) Hanging-drop experiment
In the hanging-drop experiment (see Fig.), a drop composed of a mixture of protein and buffer solution is equilibrated against a larger reservoir of buffer solution. For this experiment 24 different buffers at different pH values are used, whereby one can visualize the dependence of protein aggregation on the buffer solution in a pH range of 3.0-10.
Store the prepared crystallization plate in a suitable area at room temperature. Over the course of time, water from the drop diffuses as water vapor into the reservoir solution. This raises the concentration of the protein in the drop. After an incubation time of 24 hours, the drops are investigated under a light microscope. At this stage, different degrees of precipitation depending on the employed buffer may be noticed.
(2) Dynamic light-scattering analysis
This method provides information on the degree of aggregation of the protein within the buffer solution.
The drops which remained clear indicate sufficient solubility of the protein in the buffer and will be further investigated using dynamic light scattering.
If a narrow, monomodal size distribution and small or negligible polydispersity (< 25 %) is observed the protein sample can be transferred directly into the respective buffer solution at a concentration of 20 mM and used for crystallization screening.
(3) Additive Screen
If no buffer of Kit A yielded a monodisperse protein solution, the results can be improved by adding the additives from Kit B.
For successful crystallization screening, Jena Bioscience offers a large range of products, i.e. JBScreen Classic, JBScreen Basic, JBScreen JCSG++ and many more.
Literature citations of the JBS Solubility Kit
Pavkov-Keller et al. (2016) Structures of almond hydroxynitrile lyase isoenzyme 5 provide a rationale for the lack of oxidoreductase activity in flavin dependent HNLs. J. Biotechnol. 235:24.
BIOZ Product Citations:
Selected References:
[1] Jancarik et al. (2004) Optimum solubility (OS) screening: an efficient method to optimize buffer conditions for homogeneity and crystallization of proteins. Acta Cryst. D 60:1670.