I'd like to measure skeletal muscle glycogen content in mouse muscle samples. Does anyone know and efficient protocol or available commercial kit to measure from small amount of samples?
I am not aware of any quick method, as in using a probe or a dipstick! ;)
The old standby method for glycogen assay (Passonneau and Lauderdale, Analytical Biochemistry 60 (1974), 405-412 ) is still a commonly used method. It relies on acid hydrolysis of glycogen into glucose, followed by its conversion into glucose-6-phosphate by hexokinase, which is then converted to 6-phosphogluconic acid by G-6-P dehydrogenase (G-6-PDH) in the presence of NADP leading to a quantitative conversion to NADPH based on available G-6-P. The conversion of NADP to NADPH provides a measure of quantity by spectrophotometer (or in some cases by fluorescence).
If you are not experienced in linked enzyme assays where muiltiple enzymes are employed and certain amount of optimization of proportions and optimization of conditions becomes necessary, you may consider a ready-made kit. There are several companies that sell such kits (not very cheaply), for example:
http://www.abcam.com/glycogen-assay-kit-ab65620.html
https://www.caymanchem.com/pdfs/700480.pdf
There are a few precautions you must follow. Due to endogenous enzymes and cofactors, if the tissue sample is not handled carefully, glycogen could degrade to varying degree, which will create a large error in your measurements.
The best way is to snap freeze the the tissue sample in liquid nitrogen, powder it in a mortar pastel (pre-chilled) in LN2 and then resuspend in homoginization buffer if assay is done immediately or store it at -80ºC (or in LN2 freezer) until assay is performed. This aspect of complexity does not change irrespective of whether you mix your own reagents or use a kit.
Best...
In January 2010 – Dr’s. John Hill and Inigo San Millan
developed an ultrasound technique to detect changes in
muscle glycogen content with ultrasound. In June 2014, Dr. Hill and Inigo San Milan completed the validation study comparing muscle biopsy to ultrasound. They used High frequency ultrasound scans using
MuscleSound® technology to measure and quantify
glycogen.
Dear colleague u ca prepare a histological section from skeletal muscle ; then use the spesific stain for glycogen like best carmine or PAS stain . After staining u can asses the amount of glycogen by image analyzer program
Amal,
While what you suggest is a possibility, it is not advisable for the following reasons:
1. Not specific enough for glycogen
2. Not accurate enough for reliable quantitation
3. More time consuming than conventional quantitative glycogen measurement
Dear Sezin,
It is possible to determine glycogen accurately in cryostat sections, see Vollestad et al Acta Physiol Scand 1984, 122(4),433-441 and van der Laarse et al Biotech Histochem 1992, 67(5), 303-308. The advantage of these methods is that glycogen content can be related to fiber type
I agree with Tausif, and I can also add that PAS is not specific for glycogen, rather for aldohexoses, nor color intensity is stoichiometric, as I learned from this discussion: https://www.researchgate.net/post/Is_PAS_stain_specific_for_glycogen/1
Thank you all, especially Dr Alam, as you mentioned traditional method gave poor results probably because of the enzyme activity, but I purchased a ready Sigma-Aldrich kit which gave out reasonable results
There are available kits from sigma for example but they are super expensive (~$600 / 100 assays). However, you can do a PCA extraction on the muscle then do glycogen digestion with amyloglucosidase (40U) at 37 C for 2 h. Then run a glucose assay. You will need to run background glucose first from an allequate of the sample. Glucose after digestion - glucose before digestion = [glycogen]
Reference Bergmeyer (1983).
You can try 2 methods:
1. Glycogen Assay with HCl
2. Glycogen Assay with KOH
Glycogen kits are efficient but expensive. You can try the method of Bergmeyer et al 1983. Its a bit slow and labour intensive and the incubation takes 3 h at 37⁰C but you can do a lot of samples at once. The cost is minimal. Good luck.
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