Epitope Tag Antibody Sampler Kit  

This product is assembled as a kit. See attached protocol or CofA for further details.

Anti-6X His epitope tag polyclonal antibody (600-401-382) detects His-tagged recombinant proteins by western blot. The blot was blocked with 3% BSA in TBST for 45 min at RT. Antibody was incubated with blot at a 1:1,000 dilution in TBST with 3% BSA for 1 hour at RT. Detection occurred using HRP Gt-a-Rabbit IgG (p/n 611-103-127) diluted 1:80,000 in blocking buffer (p/n MB-070) for 30 min at RT. Lane 1 was loaded with 12-Epitope Tag Protein Marker Lysate (p/n MB-301-0100) which has the His epitope tag incorporated through a C-terminal linkage (~42 kDa). Lane 2 was loaded with His-SUMO-GFP recombinant protein which has the His epitope tag incorporated through an N-terminal linkage (~40 kDa). A 4-20% gradient gel was used to resolve the protein by SDS-PAGE. Proteins were transferred to nitrocellulose using standard methods. Molecular weights were estimated by comparison to standards (lane M).

Rockland Affinity Purified anti FLAG？ Polyclonal Antibody (600-401-383) detects both C terminal linked and N terminal linked FLAG？ tagged recombinant proteins by western blot. This antibody was used at a dilution of 1:2,500 to detect 1.0 ?g of recombinant protein containing either the FLAG？ epitope tag linked at the carboxy (C) or the amino (N) terminus of the recombinant protein. A 4-20% gradient gel was used to resolve 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 1:10,000 dilution of IRDyeR 800 conjugated Gt-a-Rabbit IgG (H&L) MX10 (code 611-132-122) for 30 min at room temperature. LICOR's OdysseyR Infrared Imaging System was used to scan and process the image. Other detection systems will yield similar results.

Rockland anti GFP polyclonal antibody (600-401-215) was used to detect GFP protein. Wild type GFP (0.1 μg) was used to spike 30 μg of a HeLa whole cell lysate. This antibody detects a 27 kDa band corresponding to the epitope tag GFP. A 4-20% Tris-Glycine gradient gel was used for SDS-PAGE. The protein was transferred to nitrocellulose using standard methods. After blocking with 5% BLOTTO in PBS, the membrane was probed overnight at 4° C with the primary antibody diluted in 5% BLOTTO to 1:1,000, followed by washes and reaction with a 1:10,000 dilution of IRDyeR 800 conjugated Goat-a-Rabbit IgG [H&L] MX10 (611-132-122). IRDyeR 800 fluorescence image was captured using the OdysseyR Infrared Imaging System developed by LI-COR. IRDye is a trademark of LI-COR, Inc. Other detection systems will yield similar results.

Rockland's anti-GST polyclonal antibody (600-101-200) in western blot shows detection of recombinant GST (indicated by band at ~ 28 kDa). The SDS-PAGE contained approximately 0.2 μg of rGST loaded on to a 4-20% gradient gel for separation. After electrophoresis, the gel was transferred to nitrocellulose and blocked with “Blocking Buffer for Fluorescent Western Blotting” p/n MB-070 in TBS for 1h at RT. The membrane was probed with anti-GST antibody at a 1:2,000 dilution in blocking reagent, overnight at 4° C. For detection DyLight？800 conjugated Donkey-a-Goat IgG (p/n 605-745-002) was used at a 1:20,000 dilution (in blocking reagent) for 30 min at 25° C. Fluorescent data was collected on a LICOR Odyssey instrument.

Western Blot of HRP anti-Goat IgG antibody (605-4313) showing detection of 50ng of goat IgG (lane 1) but not human IgG (lane 2). Samples were separated by 4-20% SDS-PAGE under reducing conditions and transferred to nitrocellulose membrane. The blot was blocked overnight at 4° C in 5% BSA in TBS. A 1:5,000 dilution of antibody in Blocking Buffer for Fluorescent Western Blotting (p/n MB-070) was used to probe the membrane at room temperature for 1 h. The image was developed using Chemiluminescent FemtoMax？ Super Sensitive HRP Substrate (p/n Femtomax-020) for one minute.