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51253

Atto 633 Protein Labeling Kit

BioReagent, suitable for fluorescence

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About This Item

UNSPSC Code:
12352200
EC Number:
200-664-3
NACRES:
NA.32
Storage temp.:
2-8°C
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product line

BioReagent

Quality Segment

manufacturer/tradename

ATTO-TEC GmbH

fluorescence

λex 633 nm; λem 661 nm in 0.1 M phosphate buffer, pH 7.0 (recommended)

suitability

suitable for fluorescence

storage temp.

2-8°C

General description

This kit contains sufficient amounts of reactive dye, buffers and protein purification sets for performing five labeling reactions (1 mg protein each) and for the subsequent purification of the labeled protein.

Application

Atto 633 is a red-emitting fluorescence label with strong absorption, high quantum yield (64%), high photostability, good water solubility, and very little triplet formation. This label is optimized for use with diode laser excitation at 633 nm and characterized by high photostability.

Legal Information

This product is for Research use only. In case of intended commercialization, please contact the IP-holder (ATTO-TEC GmbH, Germany) for licensing.

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This Item
765085114668616
fluorescence

λex 633 nm; λem 661 nm in 0.1 M phosphate buffer, pH 7.0 (recommended)

fluorescence

λex 647 nm; λem 661 nm in 0.1 M phosphate buffer, pH 7.0 (recommended)

fluorescence

λex 544 nm; λem 576 nm in 0.1 M phosphate buffer, pH 7.0 (recommended)

fluorescence

λex 594 nm; λem 625 nm in 0.1 M phosphate buffer, pH 7.0 (recommended)

manufacturer/tradename

ATTO-TEC GmbH

manufacturer/tradename

ATTO-TEC GmbH

manufacturer/tradename

ATTO-TEC GmbH

manufacturer/tradename

ATTO-TEC GmbH

Quality Level

100

Quality Level

100

Quality Level

100

Quality Level

100

storage temp.

2-8°C

storage temp.

2-8°C

storage temp.

2-8°C

storage temp.

−20°C

product line

BioReagent

product line

BioReagent

product line

BioReagent

product line

BioReagent

suitability

suitable for fluorescence

suitability

suitable for fluorescence

suitability

suitable for fluorescence

suitability

suitable for fluorescence


Storage Class

10 - Combustible liquids

wgk

WGK 3

flash_point_f

188.6 °F - closed cup

flash_point_c

87 °C - closed cup



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Questions

  1. I have an antibody dissolved in a PBS buffer that I want to label using the ATTO633 kit. Do I have to use the bicarbonate buffer for the labeling reaction or can I use the antibody solution in PBS? I am afraid the bicarbonate buffer will harm antibody.

    1 answer
    1. The carbonate buffer is provided with the ATTO643 labeling kit because the reaction of NHS-esters with lysines is most effective at around pH 8.3. At pH 7.4, most lysines are protonated and not available for the reaction. Although (de-)protonation is an equilibrium reaction, allowing all free lysines to eventually be labeled at pH 7.4, the process will be slower and less efficient. Additionally, NHS-esters slowly hydrolyze in aqueous buffers, so a longer reaction time results in more NHS-ester decay.

      If the protein tolerates high pH but not the carbonate buffer, another buffer can be used and adjusted to pH 8.3. A HEPES buffer has been successfully used for this purpose, as it maintains sufficient buffer capacity at pH >8. PBS can be adjusted to pH 8.3, but this is outside its buffer capacity, requiring careful monitoring of pH fluctuations. Refer to the buffer chart in Figure 3: https://www.sigmaaldrich.com/technical-documents/technical-article/protein-biology/gel-electrophoresis/reproducibility-with-biological-buffers. Avoid buffers containing primary amines, such as TRIS, as they will compete with lysines and reduce reaction yield.

      A pH of 8.0 might be sufficient with otherwise identical reaction conditions and is tolerated by many proteins.

      If using PBS at pH 7.4, the reaction will occur more slowly. Instead of the one-hour reaction time in the protocol, label for three hours or overnight. Increasing the dye concentration is also an option. The kit provides enough ATTO643-NHS to use twice the concentration or more. Conduct a test reaction, measure the degree of labeling (DOL), and adjust reaction time and/or dye concentration if necessary. A starting point is a 3-hour reaction time at room temperature with 1.5x the dye amount recommended in the protocol.

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