Protein Carbonyls - Biotin hydrazide is a chemical probe that specifically reacts with carbonyl groups (aldehydes and ketones) introduced into proteins by oxidative stress (protein carbonylation). These carbonyls are formed on multiple amino acid residues including histidine, lysine, cysteine, arginine, threonine, and proline [1]
.
- The reaction forms a hydrazone bond with the carbonyl groups, which can be stabilized by reduction with sodium cyanoborohydride [1]
.

Glycoproteins and glycolipids can be biotinylated using the biotinylation reagent biotin hydrazide [1].

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Labeling of Oxidized Proteins: Biotin hydrazide is used to derivatize protein carbonyls in isolated mitochondria or other protein samples. After labeling, biotinylated proteins can be detected using streptavidin conjugated to horseradish peroxidase (HRP) in Western blot assays. This method allows for the visualization and comparison of protein carbonylation levels under different conditions (e.g., aging) using 2-dimensional gel electrophoresis (IEF/SDS-PAGE) followed by Western blotting [1]
.
- Demonstration of Age-Dependent Carbonylation: Using the described protocol with biotin hydrazide, the authors demonstrated an age-dependent increase in protein carbonylation in rat testis mitochondria. Representative 2D Western blot images showed that many proteins exhibited increased carbonyl content with age, while a few proteins (e.g., spot number 7) showed a decrease, indicating the utility of this probe for studying oxidative stress in aging [1]
.

[1]. Two-dimensional gel electrophoretic detection of protein carbonyls derivatized with biotin-hydrazide. J Chromatogr B Analyt Technol Biomed Life Sci. 2016 Apr 15;1019:128-31.

Chemical Identity: Biotin hydrazide (Sigma catalog number B7639, MW: 258.34) is a small molecule probe containing a biotin moiety and a hydrazide group that reacts with carbonyls. It is typically prepared as a 60 mM stock solution in DMSO and stored at -80°C for up to 6 months [1]
.
- Purpose in Protein Oxidation Studies: Protein carbonyls are widely used biomarkers for oxidative stress during aging and in age-related diseases such as stroke, diabetes, and neurodegenerative diseases. Biotin hydrazide offers an alternative to the traditional DNPH method, avoiding issues with variable antibody affinity and non-specific detection [1]
.
- Reaction Mechanism: Biotin hydrazide reacts with protein carbonyl groups to form a hydrazone bond. This bond can be stabilized by reduction with sodium cyanoborohydride (NaCNBH₃). The resulting biotinylated proteins can then be detected with high sensitivity using streptavidin-HRP conjugates [1]
.
- Alternative Probes: The text mentions other similar probes including N'-aminooxymethylcarbonylhydrazino-D-biotin and aldehyde-reactive biotin probes that do not require a reducing agent for stabilization [1]
.
- Method Advantages: Compared to DNPH-based immunochemical detection, the biotin hydrazide/streptavidin system offers more reproducible detection and avoids problems with batch-to-batch variability of anti-DNPH antibodies [1]
.
- Sample Preparation Considerations: When using biotin hydrazide for 2D gel electrophoresis, TCA precipitation and washing steps are necessary to remove SDS which would interfere with IEF. For 1D SDS-PAGE, these steps can be omitted as the probe does not interfere with electrophoresis [1]
.
- Stabilization of Labeling: While some protocols omit sodium cyanoborohydride reduction, the authors routinely use it to stabilize the hydrazone bonds. The subsequent steps in sample preparation for IEF and SDS-PAGE involve reducing agents (DTT or β-mercaptoethanol) which may also contribute to stabilization [1]
.
- Detection Sensitivity: HRP-streptavidin can be used at dilutions up to 1:50,000 in TBST with 0.2% BSA. If signals are too strong, BSA can be replaced with 5% non-fat dried milk [1]
.
- Downstream Applications: Protein spots identified by biotin hydrazide labeling and Western blot can be matched to parallel Coomassie blue or silver-stained gels, excised, and analyzed by mass spectrometry for protein identification [1]
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- Source: Biotin hydrazide used in this protocol was obtained from Sigma [1]
.

C10H18N4O2S

258.34

258.115

66640-86-6

83872

White to off-white solid powder

1.2±0.1 g/cm3

633.1±40.0 °C at 760 mmHg

245-247 °C

336.7±27.3 °C

0.0±1.9 mmHg at 25°C

1.553

-1.24

4

4

5

17

313

3

C1[C@H]2[C@@H]([C@@H](S1)CCCCC(=O)NN)NC(=O)N2

KOZWHQPRAOJMBN-ZKWXMUAHSA-N

InChI=1S/C10H18N4O2S/c11-14-8(15)4-2-1-3-7-9-6(5-17-7)12-10(16)13-9/h6-7,9H,1-5,11H2,(H,14,15)(H2,12,13,16)/t6-,7-,9-/m0/s1

5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanehydrazide

Biotin Hydrazide (+)-Biotin Hydrazide Hydrazide biotin Biotine hydrazide Biotin-hydrazide Biotin-Hz

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~6.6 mg/mL (~25.55 mM)
H2O : ~2 mg/mL (~7.74 mM)

Solubility in Formulation 1: ≥ 1 mg/mL (3.87 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1 mg/mL (3.87 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1 mg/mL (3.87 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 2 mg/mL (7.74 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)