|USER MANUAL CM11414
CellMosaic® has designed this personalized conjugation kit to work with any protein with a MW over 20 KDa. The customer supplies its own unmodified protein. Using the kit components, the customer converts their protein to a maelimide-protein via SMCC (succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate) linker, followed by reaction of the maleimide-protein with Mertansine (DM1) to generate the conjugates (a method developed by Immunogen). One step purification typically provides the resulting protein-DM1 at greater than 95% purity.
This kit provides materials to conjugate 6.67 to 20 nmol of one (CM11414x1) or three protein samples (CM11414x3).
If you have a smaller amount of protein, a protein with MW below 20KDa, or other customization needs, please select the customization radio button and/or fill out the information above.
Requirement for protein:
- Amount: 6.67-20 nmol
- MW: >20 KDa
- Preferably >90% pure by gel electrophoresis or SEC HPLC analysis
- Protein has some lysine residues
- Name: Mertansine (DM1)
- CAS number: 139504-50-0
- Mechanism of action: Inhibits cell division by blocking the polymerisation of tubulin
Key Features of this PerKit™
- Offers a simple and easy way to label protein with DM1 with minimum exposure to the toxin
- Stable linkage
- Fast and easy preparation: 6 h preparation and <2 h hands-on time
- All reagents and supplies included for preparation and purification
- Loading is optimized based on the MW of the protein
- Options to choose tailored services at CellMosaic® after conjugation
After conjugation: you can choose to send your sample to CellMosaic® for HPLC analysis and determination of the DOL.
Schematic diagram of the workflow for preparing protein-DM1 conjugates starting with 20 nmole of protein (volume of reagents varies if the amount of IgG is < 20 nmole)
Maleimide Assay Kit (Product#: CM90002): The average maleimide groups per antibody is 4 after SMCC labeling. If necessary, you can purchase this kit separately from CellMosaic to assay the maleimide content. The kit is simple and easy to use. Approximate total assay time is 1 h.
Hydrophobic Interaction Chromatography (HIC) Buffer Set (Product#: CM02025): For PDCs prepared via surface amines of the protein, hydrophobic interaction chromatography (HIC) HPLC can be used to check if a protein is labeled or not. However, due to the highly heterogeneous nature of surface amine labeling, protein loaded with the same number of drugs (same DPR) may have slightly different hydrophobicity. For a typical DM1 PDC, a broad peak will be seen without clear separation of the peaks. CellMosaic offers HIC buffer for our customers to use with any HIC column. The CM02025 product sheet contains all of the information and methodology needed to run an HIC HPLC analysis. If you do not have access to an HPLC facility, you can send your sample to CellMosaic for analysis.
Size Exclusion Chromatography (SEC) HPLC Protein Standards (Product #: CM92004 and CM92005): DM1 is very hydrophobic. This kit is designed to minimize the aggregation and precipitation issues generally seen with DM1 labeling. However, you may still notice some solid precipitate out or PDC aggregation during the reaction. The precipitate will be removed during purification. Depending on the properties of your protein, recovery will be 30-60%. If you are concerned with the aggregation, you can use size exclusion chromatography (SEC) to check the extent of aggregation. SEC separates the conjugates by apparent molecular weight (MW) or size in aqueous solution. The larger the MW of the conjugate, the earlier it elutes. By comparing the SEC profile of unlabeled protein and the PDC, you can estimate how much aggregation is in the PDC. CellMosaic offers two SEC standards for our customers to use with any SEC column. The CM92004 product sheet contains all of the information and methodology you need to run an SEC HPLC analysis. If you do not have access to an HPLC facility, you can send your sample to CellMosaic for analysis.
ADC stabilizing PBS buffer (5x) (Product #: CM02022): CM02022 contains 5x PBS buffer and other stabilizers to prevent the hydrophobic drugs from interacting with each other during storage, which would cause the conjugates to precipitate out. Stabilization buffer also helps preserve the structure of the conjugates during lyophilization. The buffer is biocompatible and can be used directly for any in vitro and in vivo studies.