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Biocytin C2 maleimide
Biocytin maleimide readily reacts with thiol moieties of biopolymers to form thioether conjugate that is quite stable. This biocytin maleimide requires mild conjugation conditions. For example, pH of 5.5-8.5 is usually optimal for modifying cysteine residues, and air exposure of reaction solution should be minimized whenever possible to avoid the air oxidation of thiol substrates. Most conjugations are done at room temperature. However, either elevated or reduced temperature may be required for a particular labeling reaction. Reactions with this biotinylation reagent should be performed in buffers free of extraneous thiols (such as 6-mercaptoethanol, dithiothreitol and mercaptoethylamine). Proteins or peptides to be biotinylated by thiol-reactive reagents must have a free thiol group (SH) available.
Example protocol
SAMPLE EXPERIMENTAL PROTOCOL
Labeling Proteins with Biocytin C2 Malemide
- Dissolve your thiol-containing protein at concentration of 1 - 10 mg/mL (3 - 10 mg is the optimal labeling concentration) using PBS buffer (20 mM, pH 7.2).
- Dissolve the Biocytin C2 Maleimide in DMSO at a concentration of 5 - 10 mg/mL.
- Mix the Biocytin C2 Maleimide and protein solution at 2:1 molar ratio of biocytin/protein, and shake the reaction mixture at room temperature for 2 - 4 hours.
- Filter the reaction mixture through a protein spin column for 100 µg to 1 mg protein labeling reaction. If the reaction scale is larger than 1 mg, purify the conjugate using gel filtration on a properly sized Sephadex G-25 column.
- Collect the desired fractions for your immediate use or freeze dry them for future use.
Labeling Small Molecules with Biocytin C2 Malemide
- Dissolve Biocytin C2 Maleimide (10 - 15 mg/mL) and your thiol-containing molecule in DMSO at 1:1.2 molar ratio of biocytin/thiol-containing molecule.
- Stir the reaction mixture at room temperature for 2 - 4 hours.
- Purify the conjugate using HPLC (ammonium acetate/water and acetonitrile, pH 7.0).
- Collect and pool the desired fractions.
- Combine and freeze-dry the pooled fractions.
Calculators
Common stock solution preparation
Table 1. Volume of DMSO needed to reconstitute specific mass of Biocytin C2 maleimide to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
| 0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 164.303 µL | 821.517 µL | 1.643 mL | 8.215 mL | 16.43 mL |
| 5 mM | 32.861 µL | 164.303 µL | 328.607 µL | 1.643 mL | 3.286 mL |
| 10 mM | 16.43 µL | 82.152 µL | 164.303 µL | 821.517 µL | 1.643 mL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
| / | = | x | = |
Product family
| Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Correction Factor (280 nm) |
| AMCA C2 Maleimide | 346 | 434 | 19000 | 0.153 |
| EDANS C2 maleimide | 336 | 455 | 5900 | 0.107 |
| DABCYL C2 maleimide | - | - | - | 0.516 |
| XFD647 C2 Maleimide *Same Structure to Alexa Fluor™ 647 C2 Maleimide* | 650 | 671 | 239000 | 0.03 |
References
View all 51 references: Citation Explorer
The anterograde and retrograde axonal transport of biotinylated dextran amine and biocytin in the nervous system of teleosts
Authors: Xue HG, Yang CY, Ito H.
Journal: Brain Res Brain Res Protoc (2004): 106
Authors: Xue HG, Yang CY, Ito H.
Journal: Brain Res Brain Res Protoc (2004): 106
Correlative fluorescence and electron microscopy of biocytin-filled neurons with a preservation of the postsynaptic ultrastructure
Authors: Morozov Y, Khalilov I, Ben-Ari Y, Represa A.
Journal: J Neurosci Methods (2002): 81
Authors: Morozov Y, Khalilov I, Ben-Ari Y, Represa A.
Journal: J Neurosci Methods (2002): 81
Biocytin and biotin uptake into NB2a neuroblastoma and C6 astrocytoma cells
Authors: Baur B, Suormala T, Baumgartner ER.
Journal: Brain Res (2002): 111
Authors: Baur B, Suormala T, Baumgartner ER.
Journal: Brain Res (2002): 111
A new quantitative analytical method of serum biotinidase activity using biocytin as a substrate and its clinical significance in Japan
Authors: Kumasaka K, Muratsugu M, Fukui T, Kimura M, Takagi Y, Hashizume N.
Journal: Clin Chim Acta (2001): 71
Authors: Kumasaka K, Muratsugu M, Fukui T, Kimura M, Takagi Y, Hashizume N.
Journal: Clin Chim Acta (2001): 71
Biotin and biocytin uptake into cultured primary calf brain microvessel endothelial cells of the blood-brain barrier
Authors: Baur B, Baumgartner ER.
Journal: Brain Res (2000): 348
Authors: Baur B, Baumgartner ER.
Journal: Brain Res (2000): 348
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