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Hyper sensitive protein detection by Tandem-HTRF reveals Cyclin D1 dynamics in adult mouse.

Zampieri A, Champagne J, Auzemery B, Fuentes I, Maurel B, Bienvenu F - Sci Rep (2015)

Bottom Line: It is based on Homogenous Time Resolved Förster Resonance Energy Transfer (HTRF), between a lanthanide labeled donor antibody and a d2 or XL665 labeled acceptor antibody that are both raised against different epitopes of the same target.This novel approach we termed "Tandem-HTRF", can specifically reveal rare polypeptides from only a few microliters of cellular lysate within one hour in a 384-well plate format.Using this sensitive approach, we observed surprisingly that the core cell cycle regulator Cyclin D1 is sustained in fully developed adult organs and harbors an unexpected expression pattern affected by environmental challenge.

View Article: PubMed Central - PubMed

Affiliation: CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094, France.

ABSTRACT
We present here a novel method for the semi-quantitative detection of low abundance proteins in solution that is both fast and simple. It is based on Homogenous Time Resolved Förster Resonance Energy Transfer (HTRF), between a lanthanide labeled donor antibody and a d2 or XL665 labeled acceptor antibody that are both raised against different epitopes of the same target. This novel approach we termed "Tandem-HTRF", can specifically reveal rare polypeptides from only a few microliters of cellular lysate within one hour in a 384-well plate format. Using this sensitive approach, we observed surprisingly that the core cell cycle regulator Cyclin D1 is sustained in fully developed adult organs and harbors an unexpected expression pattern affected by environmental challenge. Thus our method, Tandem-HTRF offers a promising way to investigate subtle variations in the dynamics of sparse proteins from limited biological material.

No MeSH data available.


Related in: MedlinePlus

Semi-quantification by Tandem-HTRF of wildtype Cyclin D1 expression dynamics from adult organs.(a) Semi-quantification of wildtype CycD1 expression level in different adult tissues using Tandem-HTRF with Santa Cruz SC-450 antibody used as “donor” and Fisher Scientific ab1 and ab3 antibodies used as “acceptors” (right schematic). eWAT stands for epididymal white adipose tissue. Error bars = SD, n = 3. (b) Semi-quantification by Tandem-HTRF of wildtype CycD1 expression levels from homozygous Ccnd1+/+ and heterozygous Ccnd1+/− or Cdk4+/− adult lungs. Error bars = SD, n = 6. (c) Semi-quantification by Tandem-HTRF of wildtype CycD1 expression levels from homozygous Ccnd1+/+ and heterozygous Ccnd1+/− or Cdk4+/− adult kidneys. Error bars = SD, n = 3. (d) Semi-quantification by Tandem-HTRF of Ntag-CycD1 expression levels in adult Ccnd1Ntag/Ntag testis 14 hours after a single intraperitoneal injection of saline solution or Methoxy Acetic Acid at 150 mg/Kg. Error bars = SD, n = 7.
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f5: Semi-quantification by Tandem-HTRF of wildtype Cyclin D1 expression dynamics from adult organs.(a) Semi-quantification of wildtype CycD1 expression level in different adult tissues using Tandem-HTRF with Santa Cruz SC-450 antibody used as “donor” and Fisher Scientific ab1 and ab3 antibodies used as “acceptors” (right schematic). eWAT stands for epididymal white adipose tissue. Error bars = SD, n = 3. (b) Semi-quantification by Tandem-HTRF of wildtype CycD1 expression levels from homozygous Ccnd1+/+ and heterozygous Ccnd1+/− or Cdk4+/− adult lungs. Error bars = SD, n = 6. (c) Semi-quantification by Tandem-HTRF of wildtype CycD1 expression levels from homozygous Ccnd1+/+ and heterozygous Ccnd1+/− or Cdk4+/− adult kidneys. Error bars = SD, n = 3. (d) Semi-quantification by Tandem-HTRF of Ntag-CycD1 expression levels in adult Ccnd1Ntag/Ntag testis 14 hours after a single intraperitoneal injection of saline solution or Methoxy Acetic Acid at 150 mg/Kg. Error bars = SD, n = 7.

Mentions: Therefore, using a SC+ab1+ab3 Tandem-HTRF cocktail, we recorded unambiguously the signal of wildtype CycD1 protein in all of the fully developed adult organs tested (Fig. 5a). Similarly to Ccnd1Ctag/Ctagor Ccnd1Ntag/Ntag Knock In strains and depending on the adult organ, various level of wildtype CycD1 average per cell was observed (Figs 4c,d, 5a and Supplementary Fig. 11a,b). More surprisingly, we also noticed that CycD1 proportions decreased in Ccnd1+/− adult animals compared to their wildtype Ccnd1+/+ littermates (Fig. 5b,c). Tandem-HTRF could therefore measure for the first time endogenous adult CycD1.


Hyper sensitive protein detection by Tandem-HTRF reveals Cyclin D1 dynamics in adult mouse.

Zampieri A, Champagne J, Auzemery B, Fuentes I, Maurel B, Bienvenu F - Sci Rep (2015)

Semi-quantification by Tandem-HTRF of wildtype Cyclin D1 expression dynamics from adult organs.(a) Semi-quantification of wildtype CycD1 expression level in different adult tissues using Tandem-HTRF with Santa Cruz SC-450 antibody used as “donor” and Fisher Scientific ab1 and ab3 antibodies used as “acceptors” (right schematic). eWAT stands for epididymal white adipose tissue. Error bars = SD, n = 3. (b) Semi-quantification by Tandem-HTRF of wildtype CycD1 expression levels from homozygous Ccnd1+/+ and heterozygous Ccnd1+/− or Cdk4+/− adult lungs. Error bars = SD, n = 6. (c) Semi-quantification by Tandem-HTRF of wildtype CycD1 expression levels from homozygous Ccnd1+/+ and heterozygous Ccnd1+/− or Cdk4+/− adult kidneys. Error bars = SD, n = 3. (d) Semi-quantification by Tandem-HTRF of Ntag-CycD1 expression levels in adult Ccnd1Ntag/Ntag testis 14 hours after a single intraperitoneal injection of saline solution or Methoxy Acetic Acid at 150 mg/Kg. Error bars = SD, n = 7.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4622077&req=5

f5: Semi-quantification by Tandem-HTRF of wildtype Cyclin D1 expression dynamics from adult organs.(a) Semi-quantification of wildtype CycD1 expression level in different adult tissues using Tandem-HTRF with Santa Cruz SC-450 antibody used as “donor” and Fisher Scientific ab1 and ab3 antibodies used as “acceptors” (right schematic). eWAT stands for epididymal white adipose tissue. Error bars = SD, n = 3. (b) Semi-quantification by Tandem-HTRF of wildtype CycD1 expression levels from homozygous Ccnd1+/+ and heterozygous Ccnd1+/− or Cdk4+/− adult lungs. Error bars = SD, n = 6. (c) Semi-quantification by Tandem-HTRF of wildtype CycD1 expression levels from homozygous Ccnd1+/+ and heterozygous Ccnd1+/− or Cdk4+/− adult kidneys. Error bars = SD, n = 3. (d) Semi-quantification by Tandem-HTRF of Ntag-CycD1 expression levels in adult Ccnd1Ntag/Ntag testis 14 hours after a single intraperitoneal injection of saline solution or Methoxy Acetic Acid at 150 mg/Kg. Error bars = SD, n = 7.
Mentions: Therefore, using a SC+ab1+ab3 Tandem-HTRF cocktail, we recorded unambiguously the signal of wildtype CycD1 protein in all of the fully developed adult organs tested (Fig. 5a). Similarly to Ccnd1Ctag/Ctagor Ccnd1Ntag/Ntag Knock In strains and depending on the adult organ, various level of wildtype CycD1 average per cell was observed (Figs 4c,d, 5a and Supplementary Fig. 11a,b). More surprisingly, we also noticed that CycD1 proportions decreased in Ccnd1+/− adult animals compared to their wildtype Ccnd1+/+ littermates (Fig. 5b,c). Tandem-HTRF could therefore measure for the first time endogenous adult CycD1.

Bottom Line: It is based on Homogenous Time Resolved Förster Resonance Energy Transfer (HTRF), between a lanthanide labeled donor antibody and a d2 or XL665 labeled acceptor antibody that are both raised against different epitopes of the same target.This novel approach we termed "Tandem-HTRF", can specifically reveal rare polypeptides from only a few microliters of cellular lysate within one hour in a 384-well plate format.Using this sensitive approach, we observed surprisingly that the core cell cycle regulator Cyclin D1 is sustained in fully developed adult organs and harbors an unexpected expression pattern affected by environmental challenge.

View Article: PubMed Central - PubMed

Affiliation: CNRS, UMR-5203, Institut de Génomique Fonctionnelle, Montpellier, F-34094, France.

ABSTRACT
We present here a novel method for the semi-quantitative detection of low abundance proteins in solution that is both fast and simple. It is based on Homogenous Time Resolved Förster Resonance Energy Transfer (HTRF), between a lanthanide labeled donor antibody and a d2 or XL665 labeled acceptor antibody that are both raised against different epitopes of the same target. This novel approach we termed "Tandem-HTRF", can specifically reveal rare polypeptides from only a few microliters of cellular lysate within one hour in a 384-well plate format. Using this sensitive approach, we observed surprisingly that the core cell cycle regulator Cyclin D1 is sustained in fully developed adult organs and harbors an unexpected expression pattern affected by environmental challenge. Thus our method, Tandem-HTRF offers a promising way to investigate subtle variations in the dynamics of sparse proteins from limited biological material.

No MeSH data available.


Related in: MedlinePlus