RIPAb+ antibodies are evaluated using the RNA Binding Protein Immunoprecipitation (RIP) assay. Each RIPAb+ antibody set includes quantitative RT-PCR control primers (RIP Primers) to biologically validate your IP results by successfully coprecipitating a specific RNA target (where such a specific target is known). The qPCR protocol and primer sequences are provided, allowing researchers to validate RIP protocols when using the antibody in their experimental context. Each set also includes a negative control antibody to ensure specificity of the RIP reaction.
The RIPAb+ HuR set includes the HuR antibody, a negative control antibody (purified Rabbit IgG), and positive qPCR primers which amplify a 100 bp fragment of the human beta actin (ACTB) cDNA. The HuR and negative control antibodies are supplied in a scalable "per RIP" reaction size and can be used to functionally validate the precipitation HuR-associated RNAs.

Epitope: a.a. 1-13 of HuR

Immunoprecipitation from RIP lysate:
RIP lysate from MCF7 cells (2 X 10⁶ cell equivalents per IP) was subjected to immunoprecipitation using 0.5 μg of either a normal rabbit IgG or Anti-HuR antibody. Precipitated proteins were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-HuR (1.0 μg/mL).
Proteins were visualized using One-Step IP-Western kit (GenScript Cat. # L00231) (Please see figures).

Western Blot Analysis:
3T3/A31 lysate was resolved by electrophoresis, transferred to PVDF membranes and probed with HuR, hu (1:500 dilution). Proteins were visualized using a Donkey anti-Rabbit conjugated to HRP and visualized using a chemiluminescence detection system. (Please see figures).

Immunoprecipitation Analysis:
10 ug of this antibody immunoprecipitated HuR from 500 ug of 3T3/A31 RIPA lysate (Please see figures).

mmunocytochemistry Analysis:
Confocal IF analysis of HeLa, NIH/3T3 using anti-HuR (Red). Actin filaments have been labeled with AlexaFluor 488 -Phalloidin (Green). Nucleus is stained with DAPI (Blue) (Please see figures).

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

RNA binding protein Immunoprecipitation:
RIP Lysate prepared from HeLa cells (2 x 10⁷ cell equivalents per IP) were subjected to immunoprecipitation using 5 µg of either a normal Rabbit IgG or Anti-HuR antibody and the Magna RIP Kit (Cat. # 17-700).
Successful immunoprecipitation of HuR-associated RNA was verified by qPCR using RIP Primers, ACTB (Please see figures).
Please refer to the Magna RIP^® (Cat. # 17-700) or EZ-Magna RIP (Cat. # 17-701) protocol for experimental details.

35 kDa

Anti-HuR (rabbit polyclonal). One vial containing 50 μg of purified rabbit polyclonal in 100 μL of buffer containing PBS in 0.05% sodium azide with 50% Glycerol. Liquid at -20°C.

Normal Rabbit IgG. One vial containing 125 μg purified rabbit IgG in 125 μL storage buffer containing 0.1% sodium azide. Store at -20°C.

RIP Primers, ACTB. One vial containing 75 μL of 5 μM of each primer specific for human beta actin (ACTB). Store at -20°C.
FOR: TTG TTA CAG GAA GTC CCT TGC C
REV: ATG CTA TCA CCT CCC CTG TGT G

Format: Purified

Control
Included negative control rabbit IgG antibody and control primers specific for human beta actin (ATCB).

MAGNA RIP is a registered trademark of Merck KGaA, Darmstadt, Germany

UPSTATE is a registered trademark of Merck KGaA, Darmstadt, Germany