ReadiView™ Biotin: All-In-One Biotinylation & Quantification of Biotin Labeling
Fundamentals of Flow Cytometry
ReadiUse™ Lyophilized Phycobiliproteins
Buccutite™ Conjugation Technology
AssayWise Letters Vol. 8(1)
Prepare High-Performance PE, APC & Tandem Conjugates for Flow Cytometry
The use of phycobiliprotein labeled antibody conjugates has important applications in flow cytometry. Their usefulness is attributed to their individual brightness as well as their ability to create spectral separation when used as tandem dyes for multicolor analysis. A common approach for making PE, APC or tandem antibody conjugates is to use a succinimidyl 4-(N-maleimidomethyl) cyclohexane1-carboxylate (SMCC) crosslinking method.
This method uses a heterobifunctional crosslinker SMCC. SMCC crosslinkers contain N-hydroxysuccinimide (NHS) ester and maleimide groups, which allow covalent conjugation of amine (-NH2) and sulfhydryl (-SH) containing molecules. NHS esters react with primary amines at pH 7-9, while malemides react with sulfhydryl groups at pH 6.5-7.5. Because the rate of NHS esters hydrolytic degradation increases with pH, the NHS ester reaction usually occurs before or simultaneous with the maleimide reaction. For users not adept to conjugation chemistry, this may be challenging.
Although SMCC is a well-established method, it has its disadvantages. One of the major drawbacks includes poor conjugation efficiency, with a good yield at approximately 30% recovery. Secondly, SMCC requires the addition of dithiothreitol (DTT), which may significantly impact antibody immunoreactivity. Lastly, because the efficiency of SMCC-based reactions is typically low, very high input (i.e. PE and antibody) is required for successful conjugation.
Buccutite™ Technology – Simplified Workflow
An alternative to SMCC conjugation is AAT Bioquest’s novel Buccutite™ technology. Buccutite™ conjugation is a quick and simple technique for labeling PE, APC or tandem dyes to antibodies. It utilizes two separate linkers, Buccutite™ MTA and Buccutite™ FOL. These linkers are independently labeled to the phycobiliprotein and antibody of interest, and when mixed will bind strongly together to produce a bioconjugate.
To improve workflow and reduce hands-on time, Buccutite™ phycobiliprotein labeling kits are supplied with the fluorescent protein pre-activated with the Buccutite™ FOL linker. After antibody activation with Buccutite™ MTA, linker, both components are mixed and antibody conjugates are produced. The following is an overview of the protocol for Buccutite™ Rapid PE Antibody Labeling Kit (Cat#. 1310):
- Add 5 µl Reaction Buffer (Component C) into antibody (100 µl)
- Add the antibody solution into Buccutite™ MTA vial (Component B)
- Incubate at room temperature
- Remove free Buccutite™ MTA by spin column
- Mix with 50 µL Buccutite™ FOL-Activated PE (Component A)
- Incubate at room temperature
Buccutite™ vs. SMCC Conjugation
Buccutite™ offers several advantages over SMCC-based conjugation. First, Buccutite™ conjugation efficiency is relatively high, with final recovery more than double (>60%) that of SMCC conjugation. Second, the Buccutite™ reaction is efficient, conjugation can occur at low concentrations of reactants requiring a minimal sample concentration ≥0.5 mg/mL. Lastly, Buccutite™ conjugates are highly stable and can be stored at 4 °C for at least 12 months (see below for data).
In the following comparison, mouse monoclonal antibody conjugates (GXM) were prepared using Buccutite™ and SMCC conjugation. The conjugates were used to perform cell stains for flow cytometry (Figure 1). Results illustrate similarities in the positive stain and stain indexes of both conjugates, highlighting Buccutite™ as a successful alternative to SMCC.
Figure 1. Performance of GXM IgG-PE conjugates prepared with unstained control cells (blue), Mouse IgG unconjugated control (_____), Buccutite™ conjugates (Green) and SMCC conjugates (Red). HL-60 cells were stained with or without w6/32 antibody (1ug/ml) for 30min, and followed by GXM IgG-PE (5ug/ml) for 30min. The fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the PE channel.
Figure 2 illustrates the high labeling efficiency of the Buccutite™ conjugation reaction comparable to the SMCC method. More importantly, we discovered that, even without the column purification step Buccutite™ conjugation kits produced fluorescent conjugates far superior to those of unpurified SMCC conjugates. Unpurified SMCC conjugates saw a decrease in stain index by 50%.
Figure 2. Flow cytometry analysis of Jurkat cells stained with CD45-PE with different method (Buccutite™: Red; SMCC: Blue). The CD45-PE conjugate was not purified for both reaction and fluorescence signal was monitored using ACEA NovoCyte flow cytometer in the PE channel.
Buccutite™ Conjugates vs. Commercial Conjugates
PE-Cy7 (Cat# 2616) is a popular tandem dye commonly used in combination with FITC (Cat# 135), PE (Cat# 2558) and other tandem dyes for multi-parameter flow cytometry. PE-Cy7 consists of a PE phycobiliprotein labeled with the cyanine dye, Cy7 (Cat# 161). When excited by the 488 laser, PE functions as a donor transferring energy to Cy7 via Förster resonance energy transfer (FRET) to emit fluorescence at 780 nm. This difference of 292 nm between the maximal excitation and maximal emission is referred to as a Stoke’s shift. Large Stoke’s shift of tandem dyes, such as PE-Cy7, is extremely valuable when designing multicolor flow cytometry panels because it provides considerable spectral separation and minimizes crosstalk.
Figure 3. Excitation and emission spectra of PE-Cy7 (Cat# 2616).
In the following study, PE-Cy7-streptavidin conjugates prepared using Buccutite™ Rapid PE-Cy7 Tandem Labeling Kit (Cat# 1317) were compared with PE-Cy7-strepdavidin conjugates purchased from BioLegend. The performance of both conjugates was tested in HL-60 cells. Cells were stained with 1 μg/mL CD45-Biotin or mouse IgG biotin (control) and then stained with PE-Cy7-streptavidin conjugates. Results illustrate similar performance in staining, indicating Buccutite™ conjugation technology as a valuable alternative for conjugate production.
Figure 4. Performance of SA-PE/Cy7 conjugate prepared with Buccutite™ method were compared with SA-PE/Cy7 (BioLegend). HL-60 cells were stained with or without CD45 antibody (1ug/ml) for 30min, and followed by SA -PE/Cy7 (5ug/ml) for 30min. (Red peak: SA-PE/Cy7 (Buccutite™), Green peak: SA-PE/Cy7 (BioLegend).
Stability of Buccutite™ Reagents
A key advantage of Buccutite™ conjugation kits is the stability of its components. Both the Buccutite™ MTA linker and the pre-activated PE, APC or tandem dye can be lyophilized into powder form and stored at 4°C for at least 12 months without compromising product integrity. Stability was tested using CD45 antibody conjugates labeled with Buccutite™ FOL-Activated PE (Component A in Cat# 1310) and Buccutite™ FOL-Activated APC (Component A in Cat# 1311) stored at 4°C for 12 months. CD45 antibodies were pre-activated with Buccutite™ MTA (Component B in Cat# 1310 and Cat# 1311) and reacted with reconstituted Buccutite™ FOL-Activated PE and Buccutite™ FOL-Activated APC. For control, CD45 antibodies were also labeled with freshly prepared Buccutite™ FOL-Activated PE and Buccutite™ FOL-Activated APC. Jurkat cells were stained with the conjugates without further purification, and performance is shown in Figure 5. The brightness and signal-to-background ratio showed little variation between fresh Buccutite™ FOL-Activated PE/APC and Buccutite™ FOL-Activated PE/APC stored at 4°C for 12 months
Figure 5. Flow cytometry analysis of Jurkat cells stained with CD45-PE or CD45-APC prepared with Buccutite™ Kit (Cat#1310, & cat#1311). Conjugates were prepared with fresh pre-activated PE (or APC) in liquid form and lyophilized form (4oC one year old). The performance on Jurkat cell was compared using ACEA NovoCyte flow cytometer. (Green: Mouse IgG conjugate control, Red: CD45-Conjugate).
- Pre-activated phycobiliprotein and the tandems are stable for at least 12 months with the similar activity in Buccutite™ reactions, without significant changes in performance.
- Buccutite™ Ab conjugation kit is suitable to make Ab conjugate with good performance.
- The conjugates prepared with Buccutite™ technology are compatible with all other commercial conjugates, and also compatible with each other in cell surface staining for analysis and sorting.
Available Buccutite™ Labeling Kits
Buccutite™ Rapid Antibody Conjugation Kits are available for labeling antibodies and proteins with PE, APC or Tandem dyes. The phycobiliprotein or tandem dye in each kit is pre-activated with Buccutite™ FOL, and can be directly conjugated to proteins of interest activated with Buccutite MTA. The Buccutite™ FOL -activated dye readily reacts with Buccutite™ MTA-containing molecules under extremely mild neutral conditions without any catalyst required. Compared to commonly used SMCC.
Buccutite™ Rapid PE and PE-Tandem Antibody Labeling Kits
Table 1. Buccutite™ Rapid PE Antibody Labeling Kit (2 Conjugations/Kit, Each Labeling is for 100 µg Antibody).
|Cat. #||Product Name||PE Tandems|
|1310||Buccutite™ Rapid PE Antibody Labeling Kit||PE|
|1316||Buccutite™ Rapid PE-Cy5.5 Tandem Antibody Labeling Kit||PE-Cy5.5|
|1317||Buccutite™ Rapid PE-Cy7 Tandem Antibody Labeling Kit||PE-Cy7|
|1318||Buccutite™ Rapid PE-Texas Red Tandem Antibody Labeling Kit||PE-Texas Red|
|1322||Buccutite™ Rapid PE-Cy5 Tandem Antibody Labeling Kit||PE-Cy5|
Figure 6. Flow cytometry analysis of Jurkat cells stained with CD45-PE or PE Tandems prepared with Buccutite™ Kit (Cat#1310, 1316, 1317, 1318, and 1322). Jurkat cells were stained with PE conjugate and analyzed with ACEA NovoCyte flow cytometer. (Blue: Unstained cells, Green: Mouse IgG conjugate control, Red: CD45-Conjugate).
Buccutite™ Rapid APC and APC-Tandem Antibody Labeling Kits
Table 2. Buccutite™ Rapid APC Antibody Labeling Kit (2 Conjugations/Kit, Each Labeling is for 100 µg Antibody).
|Cat. #||Product Name||APC Tandems|
|1311||Buccutite™ Rapid APC Antibody Labeling Kit||APC|
|1319||Buccutite™ Rapid APC-iFluor™ 700 Tandem Antibody Labeling Kit||APC-iFluor™ 700|
|1320||Buccutite™ Rapid APC-Cy5.5 Tandem Antibody Labeling Kit||APC-Cy5.5|
|1321||Buccutite™ Rapid APC-Cy7 Tandem Antibody Labeling Kit||APC-Cy7|
Figure 7. Flow cytometry analysis of Jurkat cells stained with CD45-APC or APC Tandems prepared with Buccutite™ Kit (Cat#1311, 1319, 1320, and 1321). Jurkat cells were stained with PE conjugate and analyzed with ACEA NovoCyte flow cytometer. (Blue: Unstained cells, Green: Mouse IgG conjugate control, Red: CD45-Conjugate).
Product Ordering Information
Table 3. Product ordering information for Buccutite™ PE, APC and tandem dye antibody labeling kits.