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Hi, thanks again for posting the protocol. I have one question.After...

Yunbing Ma Jun 11, 2014

Protocol: Analysis of Mouse Liver Glycogen Content

Hi, thanks again for posting the protocol. I have one question.
After boiling the liver sample in the HCl (or NaOH), the weight is measured and water is added back in case of any evaporation. Then equal amount of NaOH or HCl is added to reneutralize the solution.
Do you test the Read more more

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Peichuan Zhang Answered Jun 12, 2014

Department of Biology, The Pennsylvania State University, USA

I'd recommend you to prepare fresh 2M NaOH, and use fresh 2M HCl for the 1st step of acid hydrolysis (dilute from the 12.1M stock solution --- please refer to http://www.sigmaaldrich.com/china-mainland/zh/chemistry/stockroom-reagents/learning-center/technical-library/reagent-concentrations.html). Yes - we used 10ul supernatant for the assay (1.0ml assay buffer with the HK enzyme). Should pH be a problem, you may consider to use less supernatant (e.g., 2ul) and increase the volume of assay solution (e.g., 1.5ml).

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Yunbing Ma Answered Jun 11, 2014

Rutgers University

Yeah..I use 1:200 in 0.05M sodium phosphate buffer (pH 7.4), and still have pH issues 90% of the time..I guess your dilution is around 1:100? (10ul supernatant+1ml assay reagent)

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Peichuan Zhang Answered Jun 11, 2014

Department of Biology, The Pennsylvania State University, USA

Hi Yunbing,

It is not necessary to measure the pH, as you will be using a rather small volume of hydrolysis product for the next step of enzyme reaction, which has a buffer for the reaction.

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