Buccutite™ Rapid PE-Cy5.5 Tandem Antibody Labeling Kit *Microscale Optimized for Labeling 100 ug Antibody Per Reaction*

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<p>AAT Bioquest offers this Buccutite™ rapid labeling kit to facilitate the PE conjugations to antibodies and other proteins such as streptavidin and other secondary reagents.  Our preactivated PE was premodified with our Buccutite™ FOL. Your antibody (or other proteins) is modified with our Buccutite™ MTA to give MTA-modified protein (such as antibody). The MTA-modified protein readily reacts with FOL-modified PE to give the desired PE-antibody conjugate in much higher yield than the SMCC chemistry. In addition, our preactivated PE reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.</p>
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2 Labelings 1316 $495


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Telephone: 1-800-990-8053
Fax: 1-408-733-1304
Email: sales@aatbio.com
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Overview

SolventDMSO
Storage Refrigerated (2-8 °C)
Minimize light exposure
Category Protein Biochemistry
General proteins
Related PE and APC
PE-Cy5.5 is a popular color used in flow cytometry. Its primary absorption peak is at 565 nm with emission peak at~700 nm. The filter sets of 682/33 nm and 695/40 nm are recommended for this tandem color. AAT Bioquest offers this Buccutite™ rapid labeling kit to facilitate the PE-Cy5.5 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Buccutite™ PE-Cy5.5 Conjugation Kit provides a robust and convenient method to conjugate your antibodies with PE. The kit includes an activated PE and reaction buffer. The conjugated antibody can be used in WB, ELISA and IHC applications. This kit is sufficient for 2 labeling reactions, each up to 100 ug of antibody. Considering the large size of PE (240 kDa), the amount of antibody used in a labeling reaction must always be less than the amount of RPE. The best ratio for any new antibody reagent must be determined by experimentation but 50-60 ug of IgG antibody for every 100 ug of RPE usually gives optimal results. Our kit provides preactivated PE-Cy5.5 to facilitate the PE-Cy5.5 tandem conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Our preactivated PE-Cy5.5 tandem is ready to conjugate, giving much higher yield than the conventionally tedious SMCC-based conjugation chemistry. In addition, our preactivated PE-Cy5.5 tandem is conjugated to a protein via its amino group that is abundant in proteins while SMCC chemistry targets the thiol group that has to be regenerated by the reduction of antibodies.




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Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.
At a glance

Protocol summary

  1. Add 5 µl Reaction Buffer (Component C) into antibody (100 µl)
  2. Add the antibody solution into Buccutite™ MTA vial (Component B)
  3. Incubate at room temperature
  4. Remove free Buccutite™ MTA by spin column
  5. Mix with 50 µL Buccutite™ FOL-Activated PE-Cy5.5 (Component A)
  6. Incubate at room temperature

Important notes
Upon receipt, store the kit at 4 oC. When stored properly, the kit should be stable for six months. Alternatively, Component B can be stored at -20°C. Do not freeze Buccutite™ FOL-Activated PE-Cy5.5 (Component A), Reaction Buffer (Component C) and Spin Column (Component D). Warm all the components and centrifuge the vials briefly before opening, and immediately prepare the required solutions before starting your conjugation. The following SOP is an example for labeling goat anti-mouse IgG antibody.

Preparation of working solution

Antibody working solution:
For labeling 100 µg antibody (assuming the target antibody concentration is 1 mg/mL), mix 5 µL (5% of the total reaction volume) of Reaction Buffer (Component C) with 100 µL of the target antibody solution. Note: If you have a different concentration, adjust the antibody volume accordingly to make ~100 µg antibody available for your labeling reaction. Note: The antibody should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4; If the antibody is dissolved in glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, or use Amicon Ultra-0.5, Ultracel-10 Membrane, 10 kDa ( Cat. # UFC501008 from Millipore) to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for antibody precipitation. Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. Note: The antibody –Buccutite™ MTA reaction efficiency is significantly reduced if the antibody concentration is less than 1 mg/mL. For optimal labeling efficiency the final antibody concentration range of 1-10 mg/mL is recommended.

Sample experimental protocol

Run Antibody-Buccutite™ MTA reaction

  1. Add the antibody solution directly into the vial of Buccutite ™ MTA (Component B), and mix them well by repeatedly pipetting for a few times or vortex the vial for a few seconds.

  2. Keep the antibody- Buccutite ™ MTA reaction mixture at room temperature for 30 - 60 minutes. Note: The antibody-Buccutite™ MTA reaction mixture can be rotated or shaken for longer time if desired.

Prepare spin column for antibody-Buccutite™ MTA purification

  1. Invert the provided spin column (Component D) several times to re-suspend the settled gel and remove any bubbles.

  2. Snap off the tip and place the column in a washing tube (2 mL, not provided). Remove the cap to allow the excess packing buffer to drain by gravity to the top of the gel bed. If column does not begin to flow, push cap back into column and remove it again to start the flow. Discard the drained buffer, and then place the column back into the Washing Tube. However, centrifuge immediately if the column is placed into a 12 x 75 mm test tube (not provided).

  3. Centrifuge for 2 minutes in a swinging bucket centrifuge at 1,000 x g (see Centrifugation Notes section) to remove the packing buffer. Discard the buffer.

  4. Apply 1-2 mL 1X PBS (pH 7.2-7.4) to the column. After each application of PBS, let the buffer drain out by gravity, or centrifuge the column for 2 minutes to remove the buffer. Discard the buffer from the collection tube. Repeat this process for 3-4 times.

  5. Centrifuge for 2 minutes in a swinging bucket centrifuge at 1,000 x g (see Centrifugation Notes section) to remove the packing buffer. Discard the buffer.

Purify the antibody-Buccutite™ MTA solution

  1. Place the column (from Step Prepare spin column for antibody-Buccutite™ MTA purification) in a clean Collecting Tube (1.5 mL, not provided). Carefully load the sample (~105 µL, from Step Antibody-Buccutite™ MTA reaction) directly to the center of the column.

  2. After loading the sample, add 5 µL of 1X PBS (pH 7.2-7.4) to make the total volume of 110 µL. Centrifuge the column for 5-6 minutes at 1,000 x g, and collect the solution that contains the desired antibody-Buccutite™ MTA solution.

Make antibody-PE-Cy5.5 conjugation

  1. Make Buccutite™ FOL-Activated PE-Cy5.5 solution by adding 50 µL ddH2O into the vial of Buccutite™ FOL-Activated PE-Cy5.5 (Component A), mix well by repeatedly pipetting for a few times or vortex the vial for a few seconds.

  2. Mix whole vial of Buccutite™ FOL-Activated PE-Cy5.5 solution into the purified antibody-Buccutite™ MTA solution (from Step Purify the antibody-Buccutite™ MTA solution), mix well and rotating the mixture for 1 hour at room temperature.

  3. The antibody-PE-Cy5.5 conjugate is now ready to use. Note: For immediate use, the antibody-PE-Cy5.5 conjugate need be diluted with the buffer of your choice. Note: For longer term storage, antibody-PE-Cy5.5 conjugate solution need be concentrated or freeze dried.

Storage of Antibody-PE-Cy5.5 Conjugate

The antibody conjugate should be stored at > 0.5 mg/mL in the presence of a carrier antibody (e.g., 0.1% bovine serum albumin). The Antibody-PE-Cy5.5 conjugate solution could be stored at 4 °C for two months without significant change when stored in the presence of 2 mM sodium azide and kept from light. For longer storage, the antibody-PE-Cy5.5 conjugates could be lyophilized and stored at ≤ –20 °C.

Centrifugation Notes

Spin column (Component D) can fit into 2 mL microcentrifuge tubes or 12 x 75 mm test tubes for sample collection during centrifugation. Use the 2 mL microtube with the columns for the initial column equilibration step.

Swinging bucket centrifuges capable of generating a minimum force of 1,000 x g are suitable for Bio-Spin column use. The gravitational force created at a particular revolution speed is a function of the radius of the microcentrifuge rotor. Consult the swinging bucket centrifuge instruction manual for the information about conversion from revolutions per minute (RPM) to centrifugal or g-force. Alternatively, use the following equation to calculate the speed in RPM required to reach the gravitational force of 1,000 x g.

RCF (x g) = (1.12 x 10-5)×(RPM)×2×r (RCF is the relative centrifugal force, r is the radius in centimeters measured from the center of the rotor to the middle of the Bio-Spin column, and RPM is the speed of the rotor).

Table 1. Avialble fluorophores at AAT Bioquest Buccutite™ Rapid Antibody Labelling Kits

Cat# Labels Ex (nm) Em (nm)
1310  PE 565 575
1322 PE-Cy5 565 674
1316 PE-Cy5.5 565 700 
1317 PE-Cy7 565 780
1318 PE-Texas Red 565 600
1311 APC 651 662
1319 APC-iFluor™ 700  651  713
1320 APC-Cy5.5  651 700
1321  APC-Cy7 651 780
1325 PerCP 482 677
Example data analysis and figures

Figure 1.

AAT Bioquest offers this Buccutite™ rapid labeling kit to facilitate the PE conjugations to antibodies and other proteins such as streptavidin and other secondary reagents.  Our preactivated PE was premodified with our Buccutite™ FOL. Your antibody (or other proteins) is modified with our Buccutite™ MTA to give MTA-modified protein (such as antibody). The MTA-modified protein readily reacts with FOL-modified PE to give the desired PE-antibody conjugate in much higher yield than the SMCC chemistry. In addition, our preactivated PE reacts with MTA-modified biopolymers at much lower concentrations than the SMCC chemistry.

Disclaimer
AAT Bioquest provides high-quality reagents and materials for research use only. For proper handling of potentially hazardous chemicals, please consult the Safety Data Sheet (SDS) provided for the product. Chemical analysis and/or reverse engineering of any kit or its components is strictly prohibited without written permission from AAT Bioquest. Please call 408-733-1055 or email info@aatbio.com if you have any questions.









References

Chromophore attachment to phycobiliprotein beta-subunits: phycocyanobilin:cysteine-beta84 phycobiliprotein lyase activity of CpeS-like protein from Anabaena Sp. PCC7120
Authors: Zhao KH, Su P, Li J, Tu JM, Zhou M, Bubenzer C, Scheer H.
Journal: J Biol Chem (2006): 8573

Excitation energy transfer from phycobiliprotein to chlorophyll d in intact cells of Acaryochloris marina studied by time- and wavelength-resolved fluorescence spectroscopy
Authors: Petrasek Z, Schmitt FJ, Theiss C, Huyer J, Chen M, Larkum A, Eichler HJ, Kemnitz K, Eckert HJ.
Journal: Photochem Photobiol Sci (2005): 1016

Single-molecule spectroscopy selectively probes donor and acceptor chromophores in the phycobiliprotein allophycocyanin
Authors: Loos D, Cotlet M, De Schryver F, Habuchi S, Hofkens J.
Journal: Biophys J (2004): 2598

Evaluation of Tolypothrix germplasm for phycobiliprotein content
Authors: Prasanna R, Prasanna BM, Mohammadi SA, Singh PK.
Journal: Folia Microbiol (Praha) (2003): 59

Isolation and characterisation of phycobiliprotein rich mutant of cyanobacterium Synechocystis sp
Authors: Prasanna R, Dhar DW, Dominic TK, Tiwari ON, Singh PK.
Journal: Acta Biol Hung (2003): 113

Co-ordinated expression of phycobiliprotein operons in the chromatically adapting cyanobacterium Calothrix PCC 7601: a role for RcaD and RcaG
Authors: Noubir S, Luque I, Ochoa de Alda JA, Perewoska I, Tandeau de Marsac N, Cobley JG, Houmard J.
Journal: Mol Microbiol (2002): 749

Phycobiliprotein genes of the marine photosynthetic prokaryote Prochlorococcus: evidence for rapid evolution of genetic heterogeneity
Authors: Ting CS, Rocap G, King J, Chisholm SW.
Journal: Microbiology (2001): 3171

Novel activity of a phycobiliprotein lyase: both the attachment of phycocyanobilin and the isomerization to phycoviolobilin are catalyzed by the proteins PecE and PecF encoded by the phycoerythrocyanin operon
Authors: Zhao KH, Deng MG, Zheng M, Zhou M, Parbel A, Storf M, Meyer M, Strohmann B, Scheer H.
Journal: FEBS Lett (2000): 9

Phycobiliprotein-Fab conjugates as probes for single particle fluorescence imaging
Authors: Triantafilou K, Triantafilou M, Wilson KM.
Journal: Cytometry (2000): 226

[Phycobiliprotein and fluorescence immunological assay]
Authors: Wu P.
Journal: Sheng Li Ke Xue Jin Zhan (2000): 82


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