Biochemistry Troubleshooting Forum

Biochemistry Troubleshooting Forum http://www.protocol-online.org/forums Wed, 08 Feb 2012 14:12:15 +0000 360 inorganic phophate http://www.protocol-online.org/forums/topic/24337-inorganic-phophate/ ANYONE done inorganic phosphors estimation? Wed, 08 Feb 2012 14:12:15 +0000 http://www.protocol-online.org/forums/topic/24337-inorganic-phophate/ Blood vessel Dihydroethidium (DHE) staining quantification using Image J http://www.protocol-online.org/forums/topic/24217-blood-vessel-dihydroethidium-dhe-staining-quantification-using-image-j/ Wei Ni, Diabetes Center at UCSF, Jan 2012
wni@diabetes.ucsf.edu

**if you can not see the pictures in this protocol properly, please check the attachment or email me for this protocol.**

I used Leica TCS SP5 Confocal Microscope for imaging, red channel for DHE signaling (pay attention to the special excitation and emission parameters) and green channel for collagen auto-fluorescence (I used the default setting for Alexa 488). Here is an example of DHE staining on mouse aorta.

Open image in Image J.
Go to Image→Color→Split Channel. You will get three images in gray scale showing signals in red (image 1), green (image 2) or blue channel. I don’t have anything in blue channel, so just ignore the split result in blue channel. As you will see, I do have auto-fluorescent signal in the red channel, which was not showing obviously in the overlay. This is why I do not recommend only capture DHE signal and use the “measure” function, which will count both DHE signal and auto-fluorescent signal. (There is an exception. If you have super strong signal, you could use very low exposure time to avoid capturing the auto-fluorescent signal.)

Image 1

Image 2
Go to Process→Image Calculator. Subtract the image green (image 2) from image red (image 1).
If the intensities of auto florescence are similar in these two channels, you will get rid off the auto fluorescence in red channel in the result (a new image will pop up, image 3). If you still have auto fluoresce in the result, just simply do the subtraction again. Make sure you do everything the same for images in all groups.

Image 3
Now we are ready to count the intensively of DHE signal. Click the result image (image 3), Go to Analyze → Measure, you will get integrated intensity for DHE signal.
You may notice, when you take images, the amount of blood vessel in each image may be different. For normalization, I count the area of auto fluorescence to represent the sample size in the image using image 2. Click image 2, go to Process→ Binary→ Make Binary (image 4). The binary step takes out the intensity variation of auto fluorescence in each image. Now, click image 4, Go to Analyze → Measure. Use the integrated intensity (represents area of auto fluorescence) for normalization.

Image 4
Calculate DHE signal / auto fluorescence signal ratio and compare each group.

Blood vessel DHE staining quantification using Image J.doc (930.5K)
Number of downloads: 1]]> Mon, 30 Jan 2012 22:37:42 +0000 http://www.protocol-online.org/forums/topic/24217-blood-vessel-dihydroethidium-dhe-staining-quantification-using-image-j/ Measuring blood glucose level http://www.protocol-online.org/forums/topic/24203-measuring-blood-glucose-level/
i know that blood glucose level can be directly measured using the Glucometer. but I would like to know the biochemical methods to measure the blood glucose level. .

Can any one help me !

How much blood i need to use for the glucose test?
How to isolate the glucose from the blood sample?
There is any titration or any biochemical methods to measure the glucose content?

please help me with ideas and protocols ..

THANKS !!!!!]]> Mon, 30 Jan 2012 02:34:37 +0000 http://www.protocol-online.org/forums/topic/24203-measuring-blood-glucose-level/ cholestrol determination in blood http://www.protocol-online.org/forums/topic/24100-cholestrol-determination-in-blood/ can any one tell me about cholestrol determiantion in blood Fri, 20 Jan 2012 19:25:40 +0000 http://www.protocol-online.org/forums/topic/24100-cholestrol-determination-in-blood/ HELP : FITC conjugated BSA http://www.protocol-online.org/forums/topic/24092-help-fitc-conjugated-bsa/
I read that FITC (without BSA conjugation) decomposes in water (ref : sigma ), .?]]> Thu, 19 Jan 2012 17:35:56 +0000 http://www.protocol-online.org/forums/topic/24092-help-fitc-conjugated-bsa/ Help - Synthesis of Peroxynitrite http://www.protocol-online.org/forums/topic/24026-help-synthesis-of-peroxynitrite/
I am doing a research project on antioxidant activities of plant extracts. I am trying to synthesize peroxynitrite for the peroxynitrite free radical scavening assay and I have been unable to do so.

I used a method similar to the one described in the paper 'Antoxidant and free radical scavening activity of Spondias pinnata' written by Bibhabasu Haszra, Santanu Biswas and Nripendranath Mandal.

We mixed:
- 5ml of acidified (0.6 Hcl) 0.7M H2O2
- 5ml NaNo2
on an ice bath for 1 second.

We then added 5ml of ice-cold 1.2M NaOH and treated the solution with MnO2 (prewashed with 1.2M NaOH) to remove excess H2O2

This was then incubated -20 degrees celius overnight. It says to then collect the peroxynitrite from the top of frozen mixture. There seems like their might be a layer on top but it is not clealy clearly visible. I repeated the experiment and once again did not seem to work.

My supervisor is away on holidays so I am alone on this one for a while. If anybody has synthesised peroxynitrite before successfully some tips and suggestions would be greatly appreciated.

Thanks guys!!!]]> Mon, 16 Jan 2012 05:01:13 +0000 http://www.protocol-online.org/forums/topic/24026-help-synthesis-of-peroxynitrite/ his tagged protein http://www.protocol-online.org/forums/topic/23886-his-tagged-protein/ I have a question for you..
I have to purchase HIV-1 aspartic proteinase to perform an inhibition assay and Sigma aldrich has only the his-tagged version. Can I buy it?Is it suitable for my test?His-tag are used in the purification process or in affinity test, is it right?Can the presence of it influence my inhibition assay?

Thank you]]> Tue, 03 Jan 2012 08:58:12 +0000 http://www.protocol-online.org/forums/topic/23886-his-tagged-protein/ Enzyme estimation http://www.protocol-online.org/forums/topic/23845-enzyme-estimation/ I need to know:
-the normal concentrations and
-the normal activites

of Fructose- 1,6-Diphosphatase, Phosphofructokinase and Ghcose-6-Phosphate Dehydrogenase in non-diabetic individuals. Where can I find that? I need to know what is the normal concentration of lactate in the serum also.

Hope someone can help me.

Cheers.]]> Sun, 25 Dec 2011 06:53:07 +0000 http://www.protocol-online.org/forums/topic/23845-enzyme-estimation/ Enzyme kinetics http://www.protocol-online.org/forums/topic/23832-enzyme-kinetics/ I need some help to interpret my results...I'm working with anthrax lethal factor, which is a Zn2+ dependent metalloproteinase. I would like to calculate Ki of an inhibitor using a FRET inhibition assay.The FRET assay are run for 30 minutes at 25 °C. At the end I obtain a graph intensity vs time which presents an hump around 10 minutes.What does it mean?Can the protein have two binding site?
You can find attached the ASCII file of the graph...

thank you|!

Attached File(s)

S11.TXT (16.36K)
Number of downloads: 4

]]> Thu, 22 Dec 2011 14:06:10 +0000 http://www.protocol-online.org/forums/topic/23832-enzyme-kinetics/ HEPES buffer preparation http://www.protocol-online.org/forums/topic/23812-hepes-buffer-preparation/ I have to prepare a 20 mM HEPES buffer, using hepes free acid form and the potassium salt. How can I calculate their quantities? I think I should use pH (pH=7.4) and pKa which is equal to 7.5.
I could also use only hepes free acid form and than adjust pH using KOH or NaOH. Which protocol is better?

thank you]]> Mon, 19 Dec 2011 21:34:22 +0000 http://www.protocol-online.org/forums/topic/23812-hepes-buffer-preparation/