Monoclonal mucin 5AC antibody (45M1, Novus Biologicals, Littleton CO, USA) was conjugated to the near-infrared (NIR) dye IRDye800CW NHS ester (LI-COR Biosciences, Lincoln, NE, USA), establishing MUC5AC-IR800. The dye was conjugated to the antibody per the manufacturer’s protocol and incubated at room temperature for 2 h on a shaker plate. After incubation, the antibody-dye conjugate was added to gel-desalting columns (Thermo Fisher Scientific, Waltham, MA, USA) to remove the excess unbound dye. The final product was stored at 4 °C.
Gel desalting columns
Manufactured by Thermo Fisher Scientific
Sourced in United States
Gel desalting columns are laboratory equipment used to remove small molecules, such as salts, from protein or other macromolecule solutions. They utilize size-exclusion chromatography to separate molecules based on their size, allowing the larger macromolecules to pass through the column while retaining the smaller molecules.
Automatically generated - may contain errors
4 protocols using gel desalting columns
1
Monoclonal Antibody Conjugation to NIR Dye
2
Antibody-Fluorophore Conjugation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
6G5j mAb was obtained from B. B. Singer (Institute for Anatomy, Essen, Germany). IR800DyeCW NHS ester (LI-COR Biosciences Inc., Lincoln, NE) was conjugated to the antibody according to the manufacturer’s protocol. The combination was incubated under basic conditions at room temperature for 2 hours on a rotator plate (Fisher Scientific, Hampton, NH). Gel desalting columns were utilized for removal of unbound dye after conjugation (Thermo Fisher Scientific, Waltham, MA). The antibody-fluorophore mixture was added to the desalting columns and this was centrifuged three times at 1500 rpm for 3 minutes. The flow-through was then removed and stored in 4°C.
3
Labeling of rHopQ with Various Fluorescent Dyes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Coupling of rHopQ to phycoerythrin (PE) was performed with the lightning-link rapid PE labeling kit (Expedeon/Abcam, Berlin, Germany), to DL488 using the Lightning-Link® Rapid DyLight 488 Antibody Labeling Kit (Novus Biological), to biotin with the Mix-n-Stain biotin antibody labeling kit (Sigma) and for IR800 with the IR800DyeCW (IR800) NHS ester system (LI-COR Biosciences Inc., Lincoln, NE) according to the manufacturer's protocol. rHopQ-IR800 was incubated on a rotator plate (Fisher Scientific, Hampton, NH) under basic conditions at room temperature for 2 h. Gel desalting columns (Thermo Fisher Scientific, Waltham, MA) were used for removal of unbound dye by adding the mixture and centrifuging three times at 2115 g for 3 min. After centrifugation, the rHopQ-IR800 flow-through was removed from the column and stored at 4 °C until further use.
4
Conjugation of MUC4 Antibody with IR800
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!