Dear Sir,

You can use the method of Hassid and Abraham, 1957 for determination of glycogen content..

There are numerous kits you can purchase to measure glycogen stores. I have attached the chemical method I employ when needed. I hope this helps.

You can use the method of Seifter et al. (1950) for determination of glycogen content as follows:

Reagents:

KOH 30%

Anthrone Reagent

Con: Sulphuric Acid

Ethyl Alcohol

Standard glucose (40mg/ml)

Procedure:

The tissues were digested in boiling KOH , and glycogen was precipitated in alcohol and which was dissolved in water and heated with anthrone reagent, so as to convert glycogen into glucose. The latter gives colour reaction.

Take 3 test tubes labelled as ‘T’, ‘S’,’B’ for test, standard and blank, respectively. Take 1ml of aliquot in the test, 1ml of standard glucose solution in standard and 1 ml distilled water in blank. All test tubes were transferred to an ice bath, 4ml of anthrone reagent was carefully added and shaken thoroughly and heated in boiling water bath for exactly 4 minutes. Once again the tubes were placed in ice bath and cooled. The intensity of the green colour developed and read at 620nm. The amount of glycogen present in tissues were expressed as mg glycogen/100mg fresh tissue weight.

Thank you all for your inputs.

I used a similar method to the one Ayman suggested, but it used a phenol sulfuric acid mixture and the reaction takes place at room femp. I downsized it though to reduce toxic waste. I bet you don't need to use such a large volume as the 1950 procedure uses. If you are interested, I can look it up for you.

Dear Frank Gelder,

I appreciate you answer and read the protocol in between the lines, I have some doubt , like is it necessary to maintain the ice condition? whether the tissue is completely dissolved in KOH solution or not? What is the purpose of adding phenol? 5% phenol in which solvent?

With regards

The cooling post boiling makes handling easy and samples must be cooled to a standard temperature. An ice bath is an easy way of consistently achieving a standard temperature between samples. No the ice bath is not necessary but temperature regulation between samples is necessary. The phenol is required for colour development. I have attached the manuscript from which this method was adapted.

Dear Sir,

You can use the method of Hassid and Abraham, 1957 for determination of glycogen content..

Thanks all of you for your inputs.

I have attached a file I wish it could help.

Hello

Kindly look at the links here 

Good Luck 

http://www.bio-protocol.org/e186

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3992013/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1180476/

Recently we performed total glycogen content, please see attached paper. Sometime using less glucose in the media help produce better signal. The first ref cited by Saleh is good point to start with. You may have to condition your cells if you are using high glucose. Following was our M&M for our paper....Good luck!!!

The human liver Huh-7.5 cell lines, containing lentivirus-overexpressed mutant PHKG2 or overexpressed wild-type PHKG2, were maintained in DMEM cell culture medium containing 10% FBS. Control cell lines were the parent Huh-7.5 cell line or the Huh-7.5 cell line containing scrambled short hairpin RNA. Cells were maintained in the presence of insulin (350 mU insulin/mL) for up to 24 h to stimulate glycogen synthesis. Cells were also maintained in DMEM medium modified to contain 1.5 mmol/L glucose and no glutamine for up to 8 h to stimulate glycogen breakdown. To harvest the cells for glycogen analysis, cells maintained as monolayers on 10-cm tissue culture plates were washed twice with PBS and scraped from plates into 1.5-mL microfuge test tubes. A sample of the cell suspension was saved for protein analysis, and cells were immediately boiled. The boiled sample was centrifuged to remove the precipitated cell pellet, and glycogen in the supernatant fraction was measured by a standard procedure (8,9).

Can somebody please explain the idea of KOH saturation in the protocol? I've seen some protocol using just 30% KOH in the first step..

Thanks for all of your input above

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Article State-of-the-Art Methods for Skeletal Muscle Glycogen Analys...

regards

I have a biomarker for specific degradation of lysosomal glycogen.