532PR‐01
G-Biosciences ♦ 1-800-628-7730 ♦ 1-314-991-6034 ♦ [email protected]
A Geno Technology, Inc. (USA) brand name
Hydroxylamine·HCl (Cat. # BC80)
think proteins! think G-Biosciences www.GBiosciences.com INTRODUCTION ...... 3 ITEM(S) SUPPLIED ...... 4 STORAGE CONDITIONS ...... 4 PROPERTIES ...... 4 STRUCTURE ...... 4 IMPORTANT INFORMATION ...... 4 PROTOCOL 1: DEACETYLATION OF SATA‐MODIFIED PROTEINS ...... 5 ADDITIONAL ITEM(S) REQUIRED ...... 5 DEACETYLATION ...... 5 PROTOCOL 2: CLEAVAGE OF PROTEIN CROSS‐LINKER EGS1 ...... 5 PROTOCOL 3: PLASMID MUTAGENESIS ...... 6 ADDITIONAL ITEM(S) REQUIRED ...... 6 HYDROXYLAMINE MUTAGENESIS ...... 6 DNA PURIFICATION WITH GET™ CLEAN DNA ...... 6 REFERENCES ...... 6 RELATED PRODUCTS ...... 7
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INTRODUCTION Hydroxylamine∙HCl is a reducing agent that is routinely used for the deacetylation of SATA to form free sulfhydryls (Figure 1), for cleavage of protein cross‐linkers that contain carbonyl groups (i.e. EGS (Cat. # BC09)) and for mutagenesis of plasmid DNA.
Figure 1: Generation of free sulfhydryls with SATA and hydroxylamine.
Hydroxylamine converts aldehydes and ketones (carbonyls) to their oxime derivative in weak bases, therefore cross‐linkers and other compounds with carbonyl groups are cleavable with hydroxylamine∙HCl.
SATA and SATP are modification reagents that add a sulfhydryl group to primary amines on biomolecules. The initial modification results in the addition of an acetyl‐protected sulfur enabling storage of the biomolecule. To generate a free sulfhydryl the biomolecule is treated with hydroxylamine to remove the protecting acetyl group (see figure).
EGS and sulfo‐EGS are homobifunctional, succinimidyl ester, amine reactive crosslinkers that are resistant to cleavage by denaturants used in SDS‐PAGE conditions, but may be cleaved with hydroxylamine.
Page 3 of 8 ITEM(S) SUPPLIED Cat. # Description Size
BC80 Hydroxylamine∙HCl 25g
STORAGE CONDITIONS Shipped at ambient temperature. Upon receipt, store desiccated at room temperature.
PROPERTIES • Synonym: Hydroxylammonium chloride
• Linear formula: NH2OH∙HCl • CAS #. 5470‐11‐1 • Molecular weight: 69.49 • Form: White crystalline powder
STRUCTURE
IMPORTANT INFORMATION • Hydroxylamine∙HCl is more stable to oxidation than the free base form, however always prepare the solutions immediately before use and store the product dessicated. • Hydroxylamine∙HCl is soluble in polar solvents such as water, ethanol, methanol, glycerol and propylene glycol.
Page 4 of 8 PROTOCOL 1: DEACETYLATION OF SATA‐MODIFIED PROTEINS
Additional Item(s) Required • Reaction Buffer. We recommend our Optimizer Buffer™ III (Cat. # BKC‐06) or PBS supplemented with 5‐25mM EDTA • SATA‐modified protein (1‐10mg/ml) • Desalting columns. SpinOUT™ GT‐600, 5ml, Cat. # 786‐704 • 1X PBS supplemented with 10mM EDTA
Deacetylation 1. Immediately before use, prepare a 0.5M solution of hydroxylamine in the Reaction Buffer. Use 35mg for every 1ml Reaction Buffer. 2. Combine 1ml SATA‐modified protein with 100µl freshly prepared 0.5M hydroxylamine∙HCl. 3. Mix and incubate at room temperature for 2 hours. 4. The excess hydroxylamine can be removed by desalting. We recommend our SpinOUT™ GT‐600, 5ml columns (Cat. # 786‐704).
PROTOCOL 2: CLEAVAGE OF PROTEIN CROSS‐LINKER EGS1 1. Immediately before use, prepare 2M hydroxylamine∙HCl in phosphate buffer at pH8.5. Adjust pH back to 8.5 after dissolving the hydroxylamine∙HCl in the buffer. 2. Rapidly warm the solution to 37°C and incubate with equal volumes of cross‐linked samples for 3‐6 hours with constant rotation or mixing. 3. The excess hydroxylamine can be removed by desalting. We recommend our SpinOUT™ GT‐600, 5ml columns (Cat. # 786‐704). 4. To determine the effect of cleavage, analyze the proteins by denaturing SDS‐PAGE.
Page 5 of 8 PROTOCOL 3: PLASMID MUTAGENESIS Derived from the protocol of Rose and Fink2.
Additional Item(s) Required • Sterile Molecular Grade Water. Molecular Grade Water (Cat. # 786‐292, 786‐293) • 5M NaOH • Plasmid DNA • DNA Clean Up Columns. GET™ CLEAN DNA (Cat. # 786‐326, 786‐357)
Hydroxylamine Mutagenesis 1. Immediately before use, prepare a 1M solution of hydroxylamine by adding 0.35g hydroxylamine∙HCl to 450µl 5M NaOH and 4.55ml ice cold. Sterile molecular grade water. If necessary, adjust pH to ~6.7 and keep solution on ice. 2. Incubate 10µg plasmid DNA with 500µl hydroxylamine solution for 20 hours at 37°C in a sealed tube.
DNA Purification with GET™ Clean DNA 1. Add 5 volumes of Binding Buffer to 1 volume of the hydroxylamine:DNA sample. 2. To bind the DNA, apply the sample to the spin column in 750µl aliquots and centrifuge for 30‐60 seconds at 14,000‐16,000g. Repeat until entire sample is loaded. 3. Discard the flow‐through. Place the column back into the same collection tube. 4. Add 750µl of DNA wash to the column and centrifuge for 30‐60 seconds at 14,000‐ 16,000g. 5. Discard flow‐through and place column back in the collection tube. Centrifuge column at 14,000‐16,000g for an additional one minute. This ensures that all DNA wash is removed. 6. Place column in a clean collection tube and elute the DNA by adding 50µl prewarmed TE buffer or water to the center of the membrane in the column and incubate at room temperature for 1 minute. Centrifuge for 1 minute at 14,000‐ 16,000g. NOTE: Alternatively, the sample can be eluted twice using half the recommended volume each time (2 x 25µl). For DNA fragments or plasmids >5kb increase TE buffer incubation to 5‐10 minutes at 55‐65°C.
REFERENCES 1. Abdella et al (1979) Biochem. Biophys. Res. Comm. 87:732 2. Rose, M.D. and Fink, G.R. (1987) Cell 48:1047
Page 6 of 8 RELATED PRODUCTS Download our Protein Cross‐linkers and Protein Labeling and Conjugation Handbooks.
http://info.gbiosciences.com/complete‐protein‐cross‐linkers‐handbook/ http://info.gbiosciences.com/complete‐protein‐labeling‐conjugation‐handbook/ For other related products, visit our website at www.GBiosciences.com or contact us.
Last saved: 6/5/2013 CMH
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