RIPAb+ FLAG - RIP Validated Antibody

DYKDDDDK sequence (FLAG) containing proteins. Reacts with both N-terminal and C-terminal fusion proteins.

RNA Binding Protein Immunoprecipitation:
RIP Lysate prepared from HeLa cells (1 X 10E7 cell equivalents per IP) expressing SNRNP70-GFP-FLAG (Cat. No. 03-903) by RNA transfection was subjected to immunoprecipitation using 5 µg of either a normal mouse IgG or Anti-FLAG M2 antibody and the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700). Successful immunoprecipitation of SNRNP70-GFP-FLAG associated RNA was verified by qPCR using RIP Primers, U1 snRNA.

Immunoprecipitation:
RIP Lysate prepared from HeLa cells (1 X 10E6 cell equivalents per IP) expressing SNRNP70-GFP-FLAG (Cat. No. 03-903) by RNA transfection was subjected to immunoprecipitation using 0.5 µg of either a normal rabbit IgG, or Anti-FLAG M2 antibody. Precipitated proteins were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-FLAG M2 (1.0 µg/mL). Proteins were visualized using Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP (Cat. # AP124P) and a chemiluminescence detection system.

This RIPAb+ FLAG -RIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.

FLAG; peptide sequence DYKDDDDK

Stable for 1 year at -20°C from date of receipt. Handling Recommendations: Upon first thaw, and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance.

Anti-FLAG M2: (mouse monoclonal IgG1): One vial containing 50 μg of protein G purified antibody in 50 μL buffered aqueous solution (50% glycerol, 10 mM sodium phosphate, and 150 mM NaCl, pH 7.4). Store at -20°C. Normal Mouse IgG: One vial containing 125 μg purified mouse IgG in 125 μL storage buffer containing 0.1% sodium azide. Store at -20°C.RIP Primers, U1 snRNA: One vial containing 75 μL of 5 μM of each primer specific for the cDNA of human U1 snRNA. Store at -20°C.FOR: GGG AGA TAC CAT GAT CAC GAA GGTREV: CCA CAA ATT ATG CAG TCG AGT TTC CC

Epitope tags provide a method to localize gene products in a variety of cell types, study the topology of proteins and protein complexes, identify associated proteins, and characterize newly identified, low abundance or poorly immunogenic proteins when protein specific antibodies are not available. Tagging with FLAG may be done at the N-terminus, N-terminus preceded by a methionine residue, C-terminus, and in internal positions of the target protein. The small size of the epitope tag and its high hydrophilicity tend to decrease the possibility of interference with protein expression, proteolytic maturation, antigenicity and function. The enterokinase cleavage site allows it to be completely removed from the purified fusion proteins.

RNA Binding Protein Immunoprecipitation: RIP Lysate prepared from HeLa cells (1 X 10E7 cell equivalents per IP) expressing SNRNP70-GFP-FLAG (Cat. No. 03-903) by RNA transfection was subjected to immunoprecipitation using 5 µg of either a normal mouse IgG or Anti-FLAG M2 antibody and the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700). Successful immunoprecipitation of SNRNP70-GFP-FLAG associated RNA was verified by qPCR using RIP Primers, U1 snRNA.
Please refer to the Magna RIP (Cat. # 17-700) or EZ-Magna RIP (Cat. # 17-701) protocol for experimental details.

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.