DigesTip pepsin is a micro-bioreactor shaped as a pipette tip with a filter containing immobilized endoproteinase pepsin, able to cleaves antibodies below hinge region, generating a F(ab')2 fragment after only 2 minutes of digestion.
DigesTip pepsin exhibits great performance in terms of ease of use, digestion time and quality of results, demonstrating that it is both a powerful resource for F(ab')2 generation and protein digestion, and a first choice to highly improve productivity in any procedure requiring pepsin digestion. DigesTip pepsin is compatible with the majority of 20 µl and 200 µl single-channel and multi-channel pipettors, including: Gilson, Biohit, Rainin, Eppendorf, Brand.
The following data show how DigesTip pepsinworks with protein samples, such as Bovine Serum Albumin (BSA), and how it deals with F(ab')2 generation from antibodies (rabbit anti-BSA antibodies).
DigesTip pepsin exhibits great performance in terms of ease of use, digestion time and quality of results, demonstrating that it is both a powerful resource for F(ab')2 generation and protein digestion, and a first choice to highly improve productivity in any procedure requiring pepsin digestion. DigesTip pepsin is compatible with the majority of 20 µl and 200 µl single-channel and multi-channel pipettors, including: Gilson, Biohit, Rainin, Eppendorf, Brand.
The following data show how DigesTip pepsinworks with protein samples, such as Bovine Serum Albumin (BSA), and how it deals with F(ab')2 generation from antibodies (rabbit anti-BSA antibodies).
Bovine Serum Albumin
DigesTip preparation time: Not required
Digestion time: 2 minute
Spectrum obtained by digesting with DigesTip pepsin, for 2 minutes at room temperature, 50 ?l of a solution of reduced and alkylated Bovine Serum Albumin at the concentration of 100 ?g/ml. Digested sample was analysed by MALDI-TOF MS, obtaining 47% of coverage and 43 peptides matched.
DigesTip preparation time: Not required
Digestion time: 2 minute
Spectrum obtained by digesting with DigesTip pepsin, for 2 minutes at room temperature, 50 ?l of a solution of reduced and alkylated Bovine Serum Albumin at the concentration of 100 ?g/ml. Digested sample was analysed by MALDI-TOF MS, obtaining 47% of coverage and 43 peptides matched.
Selection of more intense Bovine Serum Albumin (BSA) matched peptides set up to 5 missed cleavage sites (considering carboxy-side of leucine, phenylalanine, alanine and glutamic acid as cleavage sites).
Western Blot of DigesTip pepsin-digested rabbit anti-Bovine Serum Albumin (BSA) antibody.
S25 µg of rabbit anti-BSA antibodies (50 µl at 0,5 mg/ml), after digestion for 2 minutes with DigesTip pepsin, were separated by electrophoresis under non-reducing conditions and transferred by Western blotting to a nitrocellulose sheet. The membrane was then blocked with 2% fish skin gelatine. Phosphate-buffered saline containing 0.05% Tween (TPBS) was used in all washing steps. Membrane with transferred antibodies was incubated with BSA (6 ml @ 6 µg/ml) for 1 hour at room temperature and the bound BSA was then revealed with a commercial anti-BSA HRP-conjugate antibody (dilution 1/3000) using a chemiluminescent substrate (ECL reagent).
S25 µg of rabbit anti-BSA antibodies (50 µl at 0,5 mg/ml), after digestion for 2 minutes with DigesTip pepsin, were separated by electrophoresis under non-reducing conditions and transferred by Western blotting to a nitrocellulose sheet. The membrane was then blocked with 2% fish skin gelatine. Phosphate-buffered saline containing 0.05% Tween (TPBS) was used in all washing steps. Membrane with transferred antibodies was incubated with BSA (6 ml @ 6 µg/ml) for 1 hour at room temperature and the bound BSA was then revealed with a commercial anti-BSA HRP-conjugate antibody (dilution 1/3000) using a chemiluminescent substrate (ECL reagent).
Lane A): BSA bound by undigested antibody.
Lane B): BSA bound by DigesTip Pepsin-digested antibodies (2-minutes digestion).
Lane B): BSA bound by DigesTip Pepsin-digested antibodies (2-minutes digestion).
DigesTip pepsin10 µl is proven for use on a variety of automated liquid handling systems including:
- Tecan FreedomEVO Workstation
- Genomic Solutions ProMS MALDI Preparation Station
- Genomic Solutions ProPrep Protein Digestion and Mass Spec Preparation System
- Gilson Quad-Z 215 Liquid Handler with Disposable Tips
- Perkin Elmer TOFPrep MALDI Spotting System
- CyBio CyBi - Well 96 Automated Pipettor.
