Buccutite™ Rapid PE Antibody Labeling Kit *Microscale Optimized for Labeling 100 ug Antibody Per Reaction*

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<p>Flow cytometry analysis of HL-60 cells stained with 1ug/ml Mouse IgG control (Green) or with 1ug/ml mouse Anti-Human HLA-ABC (W6/32 mAb)  (Red) and then followed by Goat Anti-Mouse IgG-RPE conjugate prepared with Buccutite™ Rapid RPE Antibody Labeling Kit (Cat#1310). The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the RPE channel.</p>
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Unit Size: Cat No: Price (USD): Qty:
2 Labelings 1310 $495


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

Ex/Em (nm)565/575
SolventDMSO
Storage Refrigerated (2-4 °C)
Minimize light exposure
Category Protein Biochemistry
General proteins
Related Phycobiliproteins
R-Phycoerythrin (PE) is an orange fluorescent protein which has an excitation wavelength of 565 nm and an emission wavelength of 575 nm. 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. Buccutite™ PE Conjugation Kit provides a robust and convenient method to conjugate your antibodies with PE. The kit includes a preactivated 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 PE. 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 PE usually gives optimal results. Our kit provides preactivated PE to facilitate the PE conjugations to antibodies and other proteins such as streptavidin and other secondary reagents. Our preactivated PE is ready to conjugate, giving much higher yield than the conventionally tedious SMCC-based conjugation chemistry. In addition, our preactivated PE 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.
  1. Prepare antibody 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 1. If you have a different antibody concentration, adjust the antibody volume accordingly to make ~100 µg antibody available for your labeling reaction.
    Note 2: 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 protein precipitation.
    Note 3: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well.
    Note 4: 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.

  2. 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.

  3. 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.

  4. Purify the Ab-Buccutite™ MTA solution:
    1. Place the column (from Step 3.5) in a clean Collecting Tube (1.5 mL, not provided). Carefully load the sample (~105 μL, from Step 2.2) 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 protein-Buccutite™ MTA solution.

  5. Make Ab-PE or PE Tandem conjugation:
    1. Mix whole vial of Buccutite™ FOL-Activated PE or PE Tandem (Component A) with the purified Ab- Buccutite™ MTA solution (from Step 4.2), and rotate the mixture for 1 hour at room temperature.
    2. The Ab-PE or PE Tandem conjugate is now ready to use.
      Note 1: For immediate use, the Ab-PE or PE Tandem conjugate need be diluted with the buffer of your choice.
      Note 2: The concentration of the conjugate is about 0.5~0.6 mg Ab/mL if start with 100uL 1mg/ml antibody solution.





References & Citations

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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