Guidelines for the use and interpretation of assays for monitoring autophagy.

Guidelines for the use and interpretation of assays for monitoring autophagy.
In 2008 we revealed the primary set of tips for standardizing analysis in autophagy. Since then, analysis on this subject has continued to speed up, and plenty of new scientists have entered the sphere. Our information base and related new applied sciences have additionally been increasing.
Accordingly, it is very important replace these tips for monitoring autophagy in several organisms. Numerous evaluations have described the vary of assays which have been used for this objective. However, there continues to be confusion relating to acceptable strategies to measure autophagy, particularly in multicellular eukaryotes.
A key level that must be emphasised is that there’s a distinction between measurements that monitor the numbers or quantity of autophagic components (e.g., autophagosomes or autolysosomes) at any stage of the autophagic course of vs.
people who measure flux via the autophagy pathway (i.e., the entire course of); thus, a block in macroautophagy that ends in autophagosome accumulation must be differentiated from stimuli that lead to elevated autophagic exercise, outlined as elevated autophagy induction coupled with elevated supply to, and degradation inside, lysosomes (in most larger eukaryotes and a few protists corresponding to Dictyostelium) or the vacuole (in crops and fungi).
In different phrases, it’s particularly essential that investigators new to the sphere perceive that the looks of extra autophagosomes doesn’t essentially equate with extra autophagy. In truth, in lots of instances, autophagosomes accumulate due to a block in trafficking to lysosomes with out a concomitant change in autophagosome biogenesis, whereas a rise in autolysosomes could replicate a discount in degradative exercise.
Right here, we current a set of tips for the choice and interpretation of strategies to be used by investigators who intention to look at macroautophagy and associated processes, in addition to for reviewers who want to supply practical and cheap critiques of papers which can be centered on these processes. These tips usually are not meant to be a formulaic algorithm, as a result of the suitable assays rely partially on the query being requested and the system getting used.
As well as, we emphasize that no particular person assay is assured to be probably the most applicable one in each state of affairs, and we strongly advocate using a number of assays to watch autophagy. In these tips, we think about these varied strategies of assessing autophagy and what info can, or can not, be obtained from them. Lastly, by discussing the deserves and limits of specific autophagy assays, we hope to encourage technical innovation within the discipline.
Guidelines for the use and interpretation of assays for monitoring autophagy.

Antioxidant activity applying an improved ABTS radical cation decolorization assay.

A technique for the screening of antioxidant exercise is reported as a decolorization assay relevant to each lipophilic and hydrophilic antioxidants, together with flavonoids, hydroxycinnamates, carotenoids, and plasma antioxidants. The pre-formed radical monocation of two,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS*+) is generated by oxidation of ABTS with potassium persulfate and is diminished within the presence of such hydrogen-donating antioxidants.
The influences of each the focus of antioxidant and period of response on the inhibition of the novel cation absorption are taken into consideration when figuring out the antioxidant exercise. This assay clearly improves the unique TEAC assay (the ferryl myoglobin/ABTS assay) for the willpower of antioxidant exercise in various methods.
First, the chemistry includes the direct technology of the ABTS radical monocation with no involvement of an middleman radical. Second, it’s a decolorization assay; thus the novel cation is pre-formed previous to addition of antioxidant take a look at techniques, slightly than the technology of the novel happening regularly within the presence of the antioxidant.
Therefore the outcomes obtained with the improved system could not all the time be straight comparable with these obtained utilizing the unique TEAC assay. Third, it’s relevant to each aqueous and lipophilic techniques.

Methylation-specific PCR: a novel PCR assay for methylation standing of CpG islands.

Exact mapping of DNA methylation patterns in CpG islands has change into important for understanding various organic processes such because the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human most cancers.
We describe a brand new methodology, MSP (methylation-specific PCR), which may quickly assess the methylation standing of just about any group of CpG websites inside a CpG island, unbiased of using methylation-sensitive restriction enzymes.
This assay entails preliminary modification of DNA by sodium bisulfite, changing all unmethylated, however not methylated, cytosines to uracil, and subsequent amplification with primers particular for methylated versus unmethylated DNA. MSP requires solely small portions of DNA, is delicate to 0.1% methylated alleles of a given CpG island locus, and might be carried out on DNA extracted from paraffin-embedded samples.
MSP eliminates the false optimistic outcomes inherent to earlier PCR-based approaches which relied on differential restriction enzyme cleavage to tell apart methylated from unmethylated DNA. On this examine, we reveal using MSP to establish promoter area hypermethylation modifications related to transcriptional inactivation in 4 essential tumor suppressor genes (p16, p15, E-cadherin, and von Hippel-Lindau) in human most cancers.

New colorimetric cytotoxicity assay for anticancer-drug screening.

We have now developed a fast, delicate, and cheap methodology for measuring the mobile protein content material of adherent and suspension cultures in 96-well microtiter plates. The tactic is appropriate for odd laboratory functions and for very large-scale purposes, such because the Nationwide Most cancers Institute’s disease-oriented in vitro anticancer-drug discovery display screen, which requires using a number of million tradition wells per 12 months.
Cultures fastened with trichloroacetic acid had been stained for 30 minutes with 0.4% (wt/vol) sulforhodamine B (SRB) dissolved in 1% acetic acid. Unbound dye was eliminated by 4 washes with 1% acetic acid, and protein-bound dye was extracted with 10 mM unbuffered Tris base [tris (hydroxymethyl)aminomethane] for willpower of optical density in a computer-interfaced, 96-well microtiter plate reader.
The SRB assay outcomes had been linear with the variety of cells and with values for mobile protein measured by each the Lowry and Bradford assays at densities starting from sparse subconfluence to multilayered supraconfluence. The signal-to-noise ratio at 564 nm was roughly 1.5 with 1,000 cells per properly.
The sensitivity of the SRB assay in contrast favorably with sensitivities of a number of fluorescence assays and was superior to these of each the Lowry and Bradford assays and to these of 20 different seen dyes. The SRB assay gives a colorimetric finish level that’s nondestructive, indefinitely secure, and visual to the bare eye.

Assay Buffer, 28ML

X053-28ML 28ML
EUR 123

Assay Buffer, 55ML

X053-55ML 55ML
EUR 153

Assay Buffer, 60ML

X089-60ML 60ML
EUR 109

Assay Buffer, 50ML

X100-50ML 50ML
EUR 109

Assay Buffer, 50ML

X117-50ML 50ML
EUR 143

Lactose Assay Kit

MET-5001 100 assays
EUR 432
Description: The Lactose Assay Kit measures total lactose in milk based food products or biological samples such as blood or urine. Lactose is cleaved into glucose and galactose. Glucose is then oxidized, yielding hydrogen peroxide and D-gluconic acid. The hydrogen peroxide is detected by a fluorometric probe.

Bilirubin Assay Kit

MET-5010 200 assays
EUR 479
Description: Bilirubin, a byproduct of heme breakdown, can exist conjugated to glucuronic acid (direct) and as unconjugated (indirect). The unconjugated form is found in the blood bound to albumin and is transported to the liver. Bilirubin becomes conjugated to glucuronic acid in the liver, making it more soluble and allowing for excretion into bile. High levels of bilirubin have been correlated with jaundice and Gilbert?s syndrome while low levels have been associated with cardiovascular disease and diabetes mellitus.

Pyruvate Assay Kit

MET-5029 100 assays
EUR 479
Description: Our Pyruvate Assay Kit measures pyruvate in biological samples. First, pyruvate is oxidized by pyruvate oxidase, producing hydrogen peroxide. The hydrogen peroxide is then detected at ex. 530-570 nm/em. 590-600 nm using a specific fluorometric probe. Pyruvate levels in unknown samples are determined based on the provided pyruvate standard curve.

Glycine Assay Kit

MET-5070 100 assays
EUR 450

Taurine Assay Kit

MET-5071 200 assays
EUR 508

Sarcosine Assay Kit

MET-5072 100 assays
EUR 450

Tyrosine Assay Kit

MET-5073 100 assays
EUR 450

Phospholipid Assay Kit

MET-5085 96 assays
EUR 456

Ammonia Assay Kit

MET-5086 100 assays
EUR 456

Adenosine Assay Kit

MET-5090 100 assays
EUR 508

Inosine Assay Kit

MET-5092 100 assays
EUR 508

Alanine Assay Kit

MET-5093 200 assays
EUR 508

Urea Assay Kit

STA-382 192 assays
EUR 635
Description: Cell Biolabs? Urea Assay Kit is based on the Berthelot reaction.  Urea is first degraded into ammonia and carbon dioxide, which further reacts with an alkaline developer to produce a blue-green colored product that can be measured with a standard spectrophotometric plate reader at an optical density between 580-630 nm.  Each kit provides sufficient reagents to perform up to 192 assays, including blanks, urea standards and unknown samples.

Rac1 Activation Assay

STA-401-1 20 assays
EUR 757
Description: Our Rac Activation Assays use visible agarose beads to selectively precipitate the active form of Rac1 or Rac2. The precipitated small GTPase is then detected by Western blot using a Rac1- or Rac2-specific antibody included in the kit.

Rac2 Activation Assay

STA-401-2 20 assays
EUR 757
Description: Our Rac Activation Assays use visible agarose beads to selectively precipitate the active form of Rac1 or Rac2. The precipitated small GTPase is then detected by Western blot using a Rac1- or Rac2-specific antibody included in the kit.

Cdc42 Activation Assay

STA-402 20 assays
EUR 757
Description: Our Cdc42 Activation Assays use visible agarose beads to selectively precipitate the active form of Cdc42 protein. The precipitated small GTPase is then detected by Western blot using a Cdc42-specific antibody included in the kit.

RhoA Activation Assay

STA-403-A 20 assays
EUR 757
Description: Our Rho Activation Assays use visible agarose beads to selectively precipitate the active form of RhoA, RhoB or RhoC. The precipitated small GTPase is then detected by Western blot using a RhoA-, RhoB- or RhoC-specific antibody included in the kit.

RhoB Activation Assay

STA-403-B 20 assays
EUR 757
Description: Our Rho Activation Assays use visible agarose beads to selectively precipitate the active form of RhoA, RhoB or RhoC. The precipitated small GTPase is then detected by Western blot using a RhoA-, RhoB- or RhoC-specific antibody included in the kit.

RhoC Activation Assay

STA-403-C 20 assays
EUR 757
Description: Our Rho Activation Assays use visible agarose beads to selectively precipitate the active form of RhoA, RhoB or RhoC. The precipitated small GTPase is then detected by Western blot using a RhoA-, RhoB- or RhoC-specific antibody included in the kit.

Arf1 Activation Assay

STA-407-1 20 assays
EUR 757
Description: Our Arf Activation Assays use visible agarose beads to selectively precipitate the active form of Arf1 or Arf 6. The precipitated small GTPase is then detected by Western blot using an Arf1- or Arf6-specific antibody included in the kit.

Arf6 Activation Assay

STA-407-6 20 assays
EUR 757
Description: Our Arf Activation Assays use visible agarose beads to selectively precipitate the active form of Arf1 or Arf 6. The precipitated small GTPase is then detected by Western blot using an Arf1- or Arf6-specific antibody included in the kit.

Ral Activation Assay

STA-408 20 assays
EUR 757
Description: Our Ral Activation Assay uses visible agarose beads to selectively precipitate the active form of Ral protein. The precipitated small GTPase is then detected by Western blot using a Ral-specific antibody included in the kit.

Ran Activation Assay

STA-409 20 assays
EUR 757
Description: Our Ran Activation Assay uses visible agarose beads to selectively precipitate the active form of Ran protein. The precipitated small GTPase is then detected by Western blot using a Ran-specific antibody included in the kit.

Phosphatidylcholine Assay Kit

STA-600 96 assays
EUR 519
Description: Cell Biolabs? Phosphatidylcholine Assay Kit measures the phosphatidylcholine present within serum, plasma, or tissue samples.Samples are compared to a known concentration of phosphatidylcholine standard within the 96-well microtiter plate format.  Samples and standards are incubated for 60 minutes and then read with a standard 96-well fluorometric plate reader.

Sphingomyelin Assay Kit

STA-601 96 assays
EUR 519
Description: Cell Biolabs? Sphingomyelin Assay Kit is a simple fluorometric assay that measures the amount of sphingomyelin present in plasma or serum, tissue homogenates, or cell suspensionsin a 96-well microtiter plate format.  Each kit provides sufficient reagents to perform up to 96 assays, including blanks, sphingomyelin standards and unknown samples.  Sample sphingomyelin concentrations are determined by comparison with a known sphingomyelin standard. 

Glutamate Assay Kit

STA-674 200 assays
EUR 514
Description: Glutamate is a non-essential amino acid that serves as an important neurotransmitter in the mammalian brain and has a key role in cellular metabolism. Excess glutamate levels in the brain can cause cell injury and death, leading to neurological diseases. Our Glutamate Assay Kit is a quantitative, fluorometric assay that uses glutamate specific enzymes to generate hydrogen peroxide. An ADHP probe is oxidized by hydrogen peroxide to generate fluorescent Resorufin, which correlates to the level of glutamate in the sample. Glutamate levels in an unknown sample are calculated based on a glutamate standard curve.

Hydroxyproline Assay Kit

STA-675 96 assays
EUR 514
Description: The Hydroxyproline Assay Kit is a quantitative colorimetric assay for measuring the hydroxyproline concentration in protein samples, including collagen where it is found almost exclusively.

Autophagy Assay, Red

KF17373 50 Tests
EUR 278

Histamine Assay Kit

AKR-360 96 assays
EUR 519
Description: Histamine is naturally occurring in food, with high concentrations associated with spoiled and fermented foods. Exposure to high levels of histamine through the ingestion of food can cause symptoms similar to an allergic response. Our Histamine Assay Kit detects total histamine from food samples using a colorimetric probe. Reduction of the probe yields color development proportional the histamine levels in the sample. Absorbance at 450nm is read after a one hour incubation at 37C and histamine levels are calculated based on a histamine standard curve.

Glucose Assay Kit

abx090673-1Kit 1 Kit
EUR 237
It gives a delicate measure of drug-induced cytotoxicity, is beneficial in quantitating clonogenicity, and is properly suited to high-volume, automated drug screening. SRB fluoresces strongly with laser excitation at 488 nm and might be measured quantitatively on the single-cell degree by static fluorescence cytometry.

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