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Sigma-Aldrich

RIPAb+ AUF1 - RIP Validated Antibody and Primer Set

from rabbit

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Synonym(s):
Heterogeneous nuclear ribonucleoprotein D0, AU-rich element RNA-binding protein 1
UNSPSC Code:
12352203
eCl@ss:
32160702
NACRES:
NA.32

biological source

rabbit

Quality Level

antibody form

purified immunoglobulin

clone

polyclonal

species reactivity

human, rat, mouse

manufacturer/tradename

RIPAb+
Upstate®

technique(s)

RIP: suitable
immunoprecipitation (IP): suitable
western blot: suitable

NCBI accession no.

UniProt accession no.

shipped in

dry ice

Gene Information

human ... HNRNPD(3184)

General description

A+U Rich RNA Binding Factor (AUF1) is comprised of four isoforms of 37, 40, 42, and 45 kDa. Although the role of each isoform has yet to be fully characterized, a direct correlation has been observed between each AUF1 isoform’s binding affinity and its RNA destabilizing activity toward different AREs (A + U rich element in the 3’ untranslated region), with the isoforms p37 and p42 being the most effective.
AUF1 is a bcl2 mRNA binding protein and potentially all its isoforms are able to form complexes with the bcl2 ARE. ARE mediated bcl2 mRNA downregulation during apoptosis involves AUF1 and suggest different roles for its four isoforms.
RIPAb+ antibodies are evaluated using the RNA Binding Protein Immunoprecipitation (RIP) assay. Each RIPAb+ antibody set includes a negative control antibody to ensure specificity of the RIP reaction and is verified for the co-immunoprecipitation of RNA associated specifically with the immunoprecipitated RNA binding protein of interest. Where appropriate, the RIPAb+ set also includes quantitative RT-PCR control primers (RIP Primers) to biologically validate your IP results by successfully co-precipitating the specific RNA targets, such as messenger RNAs. The qPCR protocol and primer sequences are provided, allowing researchers to validate RIP protocols when using the antibody in their experimental context. If a target specific assay is not provided, the RIPAb+ kit is validated using an automated microfluidics-based assay by enrichment of detectable RNA over control immunoprecipitation.

Specificity

AUF1 isoforms at 37, 40, 42 and 45 kDa.
Predicted cross-reactivity with mouse and rat based on sequence homology.

Immunogen

Human AUF1 purified by Ni2+ affinity column.

Application

Immunoprecipitation from RIP lysate: Representative lot data.
RIP lysate from HeLa cells (2 X 106 cell equivalents per IP) was subjected to immunoprecipitation using 0.5 µg of either a normal rabbit IgG or Anti-AUF1 antibody. Precipitated proteins (Lane 1: rabbit IgG, Lane 2: anti-AUF1) and HeLa whole cell lysate (Lane 3) were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-AUF1 antibody (1.0 µg/mL).
Proteins were visualized using One-Step IP-Western kit (GenScript Cat. # L00231) (Please see figures).

Western Blot Analysis: 0.01-1 µg/mL of a previous lot detected AUF1 in RIPA lysates from HeLa nuclear extract and A431 cells.
Research Category
Epigenetics & Nuclear Function
Research Sub Category
RNA Metabolism & Binding Proteins

RNA Binding Protein (RBP)
This RIPAb+ AUF1 -RIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.

Packaging

10 assays per set. Recommended use: ~5 μg of antibody per RIP (dependent upon biological context).

Quality

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 5 µg of Anti-AUF1 antibody and the Magna RIP® RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700).
Successful immunoprecipitation of AUF1-associated RNA was verified by qPCR using RIP Primers FOS (Please see figures).
Please refer to the Magna RIP (Cat. # 17-700) or EZ-Magna RIP (Cat. # 17-701) protocol for experimental details.

Target description

37, 40, 42 and 45 kDa

Physical form

Anti-AUF1 (Rabbit Polyclonal). One vial containing 50 μg of protein A purified rabbit IgG in 50 μL of 70% storage buffer (0.1M Tris-glycine, pH 7.4, 0.15M NaCl, 0.05% sodium azide) and 30% glycerol. Store at -20°C.

Normal Rabbit IgG. One vial containing 125 μg Rabbit IgG in 125 μL storage buffer containing 0.05% sodium azide. Store at -20°C.

RIP Primers, FOS. One vial containing 75 μL of 5 μM of each primer specific for human FOS. Store at -20°C.
FOR: GAG AGC TGG TAG TTA GTA GCA TGT TGA
REV: AAT TCC AAT AAT GAA CCC AAT AGA TTA GTT A
Format: Purified

Storage and Stability

Stable for 1 year at -20°C from date of receipt.
Handling Recommendations: Upon receipt, 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. Note: Variability in freezer temperatures below -20°C may cause glycerol containing solutions to become frozen during storage.

Analysis Note

Control
Includes negative control rabbit IgG antibody and control primers specific for human FOS.

Legal Information

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

Disclaimer

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.

WGK

WGK 2

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable


Certificates of Analysis (COA)

Search for Certificates of Analysis (COA) by entering the products Lot/Batch Number. Lot and Batch Numbers can be found on a product’s label following the words ‘Lot’ or ‘Batch’.

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Independent exponential feeding of glycerol and methanol for fed-batch culture of recombinant Hansenula polymorpha DL-1.
H Moon,S W Kim,J Lee,S K Rhee,E S Choi,H A Kang,I H Kim,S I Hong
Applied Biochemistry and Biotechnology null
Jiahui Chen et al.
Scientific reports, 6, 32189-32189 (2016-09-01)
The identification and characterization of long non-coding RNAs (lncRNAs) in diverse biological processes has recently developed rapidly. The large amounts of non-coding RNAs scale consistent with developmental complexity in eukaryotes, indicating that most of these transcripts may have functions in
Zhen-Dong Xiao et al.
Nature communications, 8(1), 783-783 (2017-10-06)
The roles of long non-coding RNAs in cancer metabolism remain largely unexplored. Here we identify FILNC1 (FoxO-induced long non-coding RNA 1) as an energy stress-induced long non-coding RNA by FoxO transcription factors. FILNC1 deficiency in renal cancer cells alleviates energy

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