Glycogen Assay Kit II (Colorimetric) (ab169558)

Reviews (1)Q&A (2)References (9)

製品の概要

  • 製品名

    Glycogen Assay Kit II (Colorimetric)
    Glycogen キット 製品一覧

  • 検出方法

    Colorimetric

  • サンプルの種類

    Tissue, Adherent cells, Suspension cells

  • アッセイタイプ

    Quantitative

  • 検出感度

    > 4 µg/ml

  • 検出範囲

    4 µg/ml - 40 µg/ml

  • 全工程の試験時間

    1h 00m

  • 種交差性

    交差種: Other species, Mammals

  • 製品の概要

    Glycogen Assay Kit II (Colorimetric) ab169558 provides a simple, fast and robust way to measure glycogen levels in biological samples.


    This glycogen assay is suitable for measuring glycogen levels in samples that contain reducing substances, which may interfere with oxidase-based assays (such as our most popular glycogen assay kit ab65620).


    In this glycogen assay protocol, glycogen is hydrolyzed into glucose, which is oxidized to form an intermediate that reduces a colorless probe to a colored product with strong absorbance at 450 nm. This high-throughput suitable assay kit can detect 4 - 40 µg/ml of glycogen in samples.


    Glycogen assay protocol summary:
    - add samples and standards to wells
    - add hydrolysis enzyme and incubate for 30 min at room temp
    - add reaction mix and incubate for 30 min at room temp
    - analyze with microplate reader

  • 特記事項

    Review our Metabolism Assay Guide to learn about assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also about how to assay metabolic function in live cells using your plate reader.

     

    Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
    It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.

  • 試験プラットフォーム

    Microplate reader

製品の特性

  • 保存方法

    Store at -20°C. Please refer to protocols.
  • 内容 ラベル 100 tests
    Development Enzyme Mix (lyophilized) Green 1 vial
    Glycogen Development Buffer 1 x 25ml
    Glycogen Hydrolysis Buffer 1 x 25ml
    Glycogen Standard (2.0 mg/ml) Yellow 1 x 100µl
    Hydrolysis Enzyme Mix (lyophilized) Blue 1 vial
    Probe 1 vial

  • 研究分野

  • 関連性

    Glycogen is the primary short term energy storage molecule in animals. It is synthesized primarily in the liver and muscle. Glycogen is a highly branched polymer of glucose molecules, connected with an alpha-1,4 linkage, branching via an alpha-1,6 linkage. Abnormal ability to utilize glycogen is found in diabetes and in several genetic glycogen storage diseases.

画像

  • Functional Studies - Glycogen Assay Kit II (Colorimetric) (ab169558)
    Functional Studies - Glycogen Assay Kit II (Colorimetric) (ab169558)

    Glycogen measured in cell lysates showing quantity (µg) per 1 mln cells.

    Samples with the concentration of 1e7 cells/mL (HeLa) and 6.6e6 cells/mL (HCT116) were used. Samples were diluted 2-6 fold.

  • Functional Studies - Glycogen Assay Kit II (Colorimetric) (ab169558)
    Functional Studies - Glycogen Assay Kit II (Colorimetric) (ab169558)

    Glycogen measured in tissue lysates showing quantity (µg) per mg of extracted protein.

    Protein concentration for samples varied from 25 mg/mL to 50 mg/mL. Samples were diluted 2-54 fold.

  • Functional Studies - Glycogen Assay Kit II (Colorimetric) (ab169558)
    Functional Studies - Glycogen Assay Kit II (Colorimetric) (ab169558)
    Standard curve: mean of duplicates (+/- SD) with background reads subtracted
  • Glycogen metabolism
    Glycogen metabolismAw WC, Towarnicki SG, Melvin RG, et al., PLoS Genet. 2018;14(11):e1007735, Fig 14a, doi:10.1371/journal.pgen.1007735. Reproduced under the creative commons license. http://creativecommons.org/licenses/by/4.0/

    Glycogen level was highest in Alstonville larvae (n = 10 biological rep/mitotype).

参考文献

ab169558 を使用した論文を発表された方は、こちらまでお知らせください。データシートに掲載させていただきます。

ab169558 は 9 報の論文で使用されています。

  • Zhang Q  et al. Lack of muscle mTOR kinase activity causes early onset myopathy and compromises whole-body homeostasis. J Cachexia Sarcopenia Muscle 10:35-53 (2019). PubMed: 30461220
  • Aubert G  et al. Deletion of Sulfonylurea Receptor 2 in the Adult Myocardium Enhances Cardiac Glucose Uptake and Is Cardioprotective. JACC Basic Transl Sci 4:251-268 (2019). PubMed: 31061927
  • Koronowski KB  et al. Defining the Independence of the Liver Circadian Clock. Cell 177:1448-1462.e14 (2019). PubMed: 31150621
  • Rovira Gonzalez YI  et al. Mss51 deletion enhances muscle metabolism and glucose homeostasis in mice. JCI Insight 4:N/A (2019). PubMed: 31527314
  • Yang Q  et al. PRKAA1/AMPKa1-driven glycolysis in endothelial cells exposed to disturbed flow protects against atherosclerosis. Nat Commun 9:4667 (2018). PubMed: 30405100
  • Granatiero V  et al. Role of p66shc in skeletal muscle function. Sci Rep 7:6283 (2017). Functional Studies ; Mouse . PubMed: 28740219
  • Iuchi H  et al. Time-dependent effects of ipragliflozin on behaviour and energy homeostasis in normal and type 2 diabetic rats: continuous glucose telemetry analysis. Sci Rep 7:11906 (2017). Functional Studies ; Rat . PubMed: 28928461
  • Peng X  et al. Thioredoxin reductase 1 suppresses adipocyte differentiation and insulin responsiveness. Sci Rep 6:28080 (2016). Mouse . PubMed: 27346647
  • Perry MC  et al. Estrogen-related receptor-a coordinates transcriptional programs essential for exercise tolerance and muscle fitness. Mol Endocrinol 28:2060-71 (2014). Mouse . PubMed: 25361393

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Measurement of Glycogen concentration in zebrafish embryos

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1) Dechorionate the embryos mechanically at 24 hpf
2) Remove the yolk
• Protocol for deyolking:
- Put the 10 dechorionated embryos in an eppendorf tube (1.5 ml) using a plastic
pipette, and place on ice to sedate the embryos.
- Take the eppendorf tube containing the embryos from ice, remove as much fluid (fish water/egg water) as possible and add deyolking buffer (*).
- Remove the yolks by triturating with a 200 µl pipette tip.
- Let the embryos float down to the bottom of the tube, and remove as much
deyolking fluid as possible.
- Rince twice with ice cold Ringer’s solution.
- Remove as much liquid as possible and put closed eppendorf tube in liquid nitrogen for 10 min, and store at -80ºC.
Or: you can proceed immediately with the protocol for Glycogen Assay Kit II analysis.
1. Glycogen Standard Curve:
Dilute Glycogen Standard to 0.2 mg/ml (0.2 µg/μl) by adding 10 µl of 2 mg/ml Glycogen Standard to 90 µl dH2O, mix well. Add 0, 2, 4, 6, 8 and 10 µl of 0.2 mg/ml Glycogen Standard into series of wells in 96 well plate to generate 0, 0.4, 0.8, 1.2, 1.6
and 2 µg/well Glycogen Standard. Adjust volume to 50 µl per well with Glycogen Hydrolysis Buffer.
2. Sample preparation:
10 zebrafish embryos should be rapidly homogenized with 200 µl ddH2O for 10 minutes on ice. Boil the homogenates for 10 minutes to inactivate enzymes. Centrifuge at 14500 rpm for 15 minutes and remove insoluble material. Collect the supernatant. Supernatant is ready to be assayed. Add 10-50 µl samples (~50 µg) into a 96 well plate and bring the volume to 50 µl with Glycogen Hydrolysis Buffer.
3. Hydrolysis:
Add 2 µl of Hydrolysis Enzyme Mix to Standard and samples, mix well. Incubate at room temperature for 30 minutes.
Note: Don’t add Hydrolysis Enzyme Mix to the sample background control.
4. Reaction Mix:
Mix enough reagents for the number of samples and standards to be performed. For each well, prepare a total 50 µl Reaction Mix containing:

Add 48 µl of the Reaction Mix to each well containing the Standard and samples and 50 µl of Background Control Mix to background control well.
5. Measurement:
Incubate at room temperature for 30 minutes. Measure OD450 nm with a microplate reader.
6. Data Analysis
Calculation: Subtract 0 Glycogen Standard reading from all readings. Plot the Glycogen Standard curve. If background control reading is significant, subtract the background control reading from sample reading. Apply the corrected sample reading to the Glycogen Standard curve to get B µg of Glycogen in the sample.

Figure 1 (A) Column 1: ug per well Glycogen Standard, Column 2 and 4: absorbance (Abs) values from first and second lecture, Column 3 and 5: absorbance less background Column 6: media of absorbance values. (B) Glycogen Standard Curve.

7. Results
To achieve the quantity of glycogen (ug) present in our sample, we applied the formula obtained from the standard curve plot:
y = 0,0876x – 0,0828
x = (y + 0,0828) / 0,0876 = glycogen amount from Standard Curve (B)
Sample Glycogen Concentration (C) = B (ug) / Sample volume used in the reaction (ul)

Figure 2 (A) Column 2 and 4: absorbance (Abs) values from first and second lecture, Column 3 and 5: absorbance less background Column 6: glycogen values obtained applying the formula: C = B (ug) / Sample volume used in the reaction (ul).
(B) Glycogen present in zebrafish embryos at 24 hours post fertilization (hpf).

The embryos used were zebrafish from AB strain line, breeded in fish water.

(*): DEYOLKING BUFFER
To obtain 400ml di ½ Ginzburg fish Ringer solution (deyolking buffer) weigh: 1,3 g NaCl; 0,05 g KCl; 0,06 g CaCl2 or 0,08 CaCl2 2H2O; Add milliQ water almost till 400 ml; Add 0,04 g of NaHCO3; Bring to volume (400 ml) with milliQ water; Filter and store at RT.

Dr. Cinzia Bragato

Verified customer

投稿 Jan 17 2018

Question

I am interested in quantifying glycogen in liver and muscle from mice and found this kit on your website, ab169558 Glycogen Assay Kit II, I was just wondering if this is a suitable kit for analyzing homogenized tissue  from mice? And one technical question, can I the freeze the supernatant from the homogenized tissue in order to analyze it once I have collected samples from all animals? (B2 Sample preparation in the protocol)


 

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Answer

Thank you for your enquiry.

I can confirm that the kit is not species specific so it should be suitable for mouse. It has also been tested for tissue samples, so this wil also be covered by the guarantee.


https://www.abcam.com/content/abpromise-guarantee

Regarding sample preparation and storage, fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.

I hope this will be helpful.

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Question

In comparing these 2 Glycogen assay kits (Glycogen assay kit and glycogen assay kit II) I see that Assay Kit II requires no boiling step to inactivate enzymes in the sample. but Glycogen assay kit (I) does require a boiling step. Is this a mistake or is there something different about the buffers in assay Kit II that makes boiling unncecessary.

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Answer

ab65620, Glycogen Assay Kit, can detect glycogen 0.0004 to 2 mg/ml. This kit can be used in both colorimetric and fluorometric applications. In this assay, glucoamylase hydrolyzes the glycogen to glucose which is then specifically oxidized to produce a product that reacts with OxiRed probe to generate color (λmax= 570 nm) and fluorescence (Ex 535/Em 587).

Since the fluorometric version is very sensitive, this improved kit uses glycogen extraction and removes all enzyme activity by boiling to reduce glycogenolysis and the samples can be stored after this step to be assayed later.


ab169558, Glycogen Assay Kit II (Colorimetric), can detect down to around 4 µg/ml of Glycogen in samples. This kit is colorimetric only. This assay is suitable for measuring Glycogen levels in samples that contain reducing substances, which may interfere with the oxidase-based assays like that used in ab65620. In this assay, Glycogen is hydrolyzed into glucose, which is oxidized to form an intermediate that reduces a colorless Probe to a colored product with strong absorbance at 450 nm.

This is faster protocol without any glycogen extraction but not as sensitive as the fluorometric version.

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