Enzyme Immunoassay (E), Western blot / Immunoblot (WB), Frozen Sections (C), Immunocytochemistry/Immunofluorescence (ICC/IF), Immunoprecipitation (IP), ELISA (capture) (E(capture))
Background of Maltose Binding Protein Tag / MBP-Tag antibody
Epitope tags are short peptide sequences that are easily recognized by tag-specific antibodies. Due to their small size, epitope tags do not affect the tagged protein’s biochemical properties. Most often sequences encoding the epitope tag are included with target DNA at the time of cloning to produce fusion proteins containing the epitope tag sequence. This allows anti-epitope tag antibodies to serve as universal detection reagents for any tag containing protein produced by recombinant means. This means that anti-epitope tag antibodies are a useful alternative to generating specific antibodies to identify, immunoprecipitate or immunoaffinity purify a recombinant protein. The anti-epitope tag antibody is usually functional in a variety of antibody-dependent experimental procedures. Expression vectors producing epitope tag fusion proteins are available for a variety of host expression systems including bacteria, yeast, insect and mammalian cells.
Fox JD., et al. (2001). Protein Sci. 10(3): 622-30 Riggs., et al. (2000). Mol. Biotechnol. 15: 51-63
Lane1: MBP-NOL6 64kd; lane2 MBP 42kd were subjected to SDS PAGE followed by western blot with 15089-1-AP(MBP-Tag Antibody) at dilution of 1:3000
Antibody sensitivity: MBP was separated by SDS-PAGE and transferred to PVDF membranes. Immunoblots were probed with MBP antibody (6E4) Cat.-No AM00088PU-N (0.5 µg/ ml) for 1h at RT and developed by ECL (exp. time: 30 sec). Lane 1: 100ng MBPLane 2: 50ngLane 3: 25ng Lane 4: 10ng Lane 5: 5ng Lane 6: 2ng Lane 7: 1ng
Western blot analysis: Recombinant protein MBP-tagged BCDEF (Lane 1), TPD52 (Lane 2) and MAGEA3 (Lane 3) (each 20 ng) were resolved by SDS-PAGE, transferred to PVDF membrane and probed with anti-MBP (1:1000). Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP and an ECL detection system.
Anti-MBP epitope tag polyclonal antibody detects MBP-tagged recombinant proteins by western blot. Polyclonal rabbit-anti-MBP epitope tag at 0.5-1.0 µg/ml was used to detect 1.0 ug of recombinant protein containing the MBP epitope tag. A 4-20% gradient gel was used to separate the protein by SDS-PAGE. The protein was transferred to nitrocellulose using standard methods. After blocking the membrane was probed with the primary antibody for 1 h at room temperature followed by washes and reaction with a 1:2500 dilution of IRDye(TM)800 conjugated Gt-a-Rabbit IgG [H&L] for 30 min at room temperature. LICOR's Odyssey(R) Infrared Imaging System was used to scan and process the image. Other detection systems will yield similar results.
Western Blot showing detection of Maltose Binding Protein (MBP) (0.05 µg) in Lane 2. MW markers indicated in Lane 1. Protein was run on a 4-20% gel and transferred to 0.45 µm nitrocellulose. After blocking with 1% BSA-TTBS (p/n MB-013, diluted to 1X) 30 min at 20°C Anti-MBP (RABBIT) antibody (AP09126PU) was used at 1/1000 overnight at 4°C. Anti-Rabbit IgG (GOAT) IRDye800® conjugated antibody secondary antibody was used at 1/20,000 in Blocking Buffer for Fluorescent Western Blotting for 30 min at 20°C and imaged on the LiCor Odyssey imaging system. A band is present at the correct molecular weight, ~42 kDa, the other bands present are recombinant MBP breakdown.