RIPAb+ PUM1 Antibody

Within the mammalian genome are two genes belonging to a family of housekeeping genes known as PUF. These genes are known as PUM1 and PUM2. PUF proteins are characterized by having a C-terminal RNA-binding domain called the Pumilio homolog domain, which affects the stability of mRNA and translation by binding the 3’-UTR of mRNA targets. PUM plays an active role in the sustainability of mitotic stem cell proliferation and the regulation of cell development and differentiation. Very little has been discovered about the specific role for PUM1. However it has been shown to bind an mRNA sequence known as Nanos response element and to interact with RNA. PUM1 is found in heart, brain, muscle, kidney, stomach and intestine. It is not expressed in putamen, hippocampus, medulla oblangata, corpus collasum, cerebellum and caudate nucleus.

~120 kDa
UniProtKB/Swiss-Prot Q14671 (PUM1_HUMAN) entry describes 2 isoforms produced by alternative splicing: Isoform 1 at ~126 kDa and Isoform 2 at ~124 kDa

KLH-conjugated linear peptide corresponding to the N-terminus of human Pumilio-1.

RIPAb+ PUM1 Antibody detects level of RIPAb+ PUM1 & has been published & validated for use in ICC, Western Blotting, RNA Binding Protein Immunoprecipitation (RIP).

Research Sub Category
Hematopoietic Stem Cells

RNA Metabolism & Binding Proteins

Western Blot Analysis:
Representative lot data.
H9 human stem cell lysate was resolved by electrophoresis, transferred to PVDF membranes and probed with Anti-Pumilio-1 (PUM1) (1:5,000 dilution).
Proteins were visualized using a Donkey Anti-Rabbit IgG conjugated to HRP and chemiluminescence detection system.
Arrow indicates Pumilio-1 (~120 kDa).
Immunocytochemistry Analysis:
Representative lot data.
Confocal fluorescent analysis of mouse and human embryonic stem cells using Anti-Pumilio-1 (PUM1) (Red). Actin filaments have been labeled with Alexa Fluor^™ 488 dye - Phalloidin (Green). Nucleus is stained with DAPI (Blue). This antibody positively stains the cytosol.

This antibody recognizes the N-terminus of Pumilio-1 (PUM1).

10 assays per set. Recommended use: ~2.5 µg (7.2 µL) of antibody per RIP (dependent upon biological context).

Components:
Anti-PUM1 (Rabbit Polyclonal). One vial containing 25 µg of affinity purified polyclonal in buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl,with 0.05% sodium azide before the addition of 30% glycerol. Store at -20°C.
Concentration: 0.35 mg/mL
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, p27 mRNA. One vial containing 75 μL of each primer (5 μM) specific for a region of the human p27 (Kip1/CDKN1B) mRNA. Store at -20°C.
FOR: CTG CAA CCG ACG ATT CTT CTA CT
REV: GGG CGT CTG CTC CAC AGA

Control
Includes normal rabbit IgG and primers specific for human p27 mRNA.

RNA Binding Protein Immunoprecipitation:
Representative lot data.
RIP Lysate prepared from HeLa cells (2 X 10E7 cell equivalents per IP) were subjected to immunoprecipitation using 2.5 µg of either a normal Rabbit IgG or 2.5 µg Anti-PUM1 antibodyand the Magna RIP^® RNA-Binding Protein Immunoprecipitation Kit (Cat. # 17-700). Successful immunoprecipitation of PUM1-associated RNA was verified by qPCR using RIP Primers p27 mRNA, (Figure 1).
Please refer to the Magna RIP (Cat. # 17-700) or EZ-Magna RIP (Cat. # 17-701) protocol for experimental details.

Concentration: Please refer to the Certificate of Analysis for the lot-specific concentration.

ALEXA FLUOR is a trademark of Life Technologies

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

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