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Questions related from Sulail Fatima
I am facing some trouble while pelleting tissue debris. I am using the attached protocol. After centrifugation and removing supernatant, I store my supernatant in -80c until bca estimation which...
18 January 2025 1,099 5 View
My 10X lysis buffer has 10% (w/v) SDS, 100 mM TRIS, 10 mM EDTA. After making 1X I add PI cocktail. I have been reading three different variations for temperature for protein extraction (boiling...
20 December 2024 6,730 6 View
I normalized my samples to 3 µg/µL and loaded 30 µg and 40 µg per lane. However, I observed that my target band appears M-shaped, even after Ponseau staining, where the higher molecular weight...
07 December 2024 3,756 6 View
I am trying to extract protein from a 3mg tissue. I used 85ul ripa and used a bead homogenizer. It’s a nervous tissue. The protein concentration were satisfactory (using bca) and I proceeded with...
19 November 2024 481 1 View
I would like to reuse my primary antibody for western blotting. I am looking for an optimal way to store. I block my membranes in 5% skim milk followed by washing and primary antibody incubation....
23 October 2024 9,693 3 View
I am running a 12 % SDS gel for my protein expression (targets are between 35 - 17 kDa). I use a mini biorad setup for gel preparation. I run at 60V for 30' followed by 100V for 1 hour and 15...
20 October 2024 3,145 4 View
I performed gelatin zymography on cardiac tissue extracts. I was expecting mmp-2 and mmp-9 bands as reported by references. Where did I go wrong? Both attachments are same gels. Just there is the...
17 January 2018 5,301 19 View
I am performing Gelatin Zymography for evaluating the activity of Gelatinases. The enzymes are separated in denaturing conditions (SDS, during electrophoresis) and then, refolded in Triton to...
17 January 2018 3,940 18 View
I extracted RNA using Riboex (Geneall) followed by Trizol method. I have 2 types of tissues: Infarcted & non-infarcted cardiac tissue ( I am planning to perform RT-qPCR). I guess I am having some...
01 January 2018 7,283 16 View
It might sound lame but I am new to it. I don't get first I must optimize the primer or check for my qpcr efficiency by serially diluting my cdna. Can anyone briefly explain how I can do these...
16 December 2017 5,151 3 View
According to this, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2907514/ Polr2a, Rpl32 and Tbp seem suitable for studying ventricular remodeling/heart failure in rats' cardiac tissue. Kindly share...
06 August 2017 8,775 7 View
Can anyone suggest which enzymes I shall use for preparing single cell suspension from cardiac tissue. I will be looking at both cell surface and intracellular antigens involving cardiomyocytes...
15 July 2017 4,823 3 View
I have been reading about various drugs inducing anti-remodeling effects in animal models (rats) of myocardial infarction. Can someone suggest why ischemia-reperfusion is not preferred in this...
16 March 2017 7,478 4 View
I am using Labchart 5 to record intra-carotid & intra-ventricular pressure. Can anyone suggest how can we obtain dp/dt minimum and maximum? After selecting the pressure waves during a specific...
07 March 2017 4,972 5 View
After arresting the rat's heart diastole, I freeze it for an hour and half. I use rat heart slicer to make sections (2 mm) for ttc staining. After staining, my most of the sections are wrinkled. I...
12 December 2016 698 2 View
I am studying MMP-2 and MMP-9 activity by gelatin zymography. The bands I have obtained are smeared up (within their lanes). Can anyone suggest possible reasons for this?
24 August 2016 4,974 9 View
A drug that has no anti-inflammatory and anti-oxidative effects. Furthermore, it shall not induce cardioprotection. Also that, I am working on opioid system the drug shall not be a opioid agonist....
28 April 2016 8,031 3 View
I am presenting an article in weekly journal club meeting and the authors have used " Masson's Trichrome Staining and Picrosirius red staining to asses fibrosis & collagen content,...
03 January 2016 6,926 7 View
I wish to study the effect of my drug on fibroblast differentiation into myofibroblasts. So far, I am unable to hold on to any marker that is specific for myofibroblasts (is not expressed by any...
14 October 2015 9,082 4 View
I need to perform gelatinase zymography (mmp2 & mmp9) and so far the best sample to be used I have come across is serum free media. I can't afford expensive growth factors for my work. Can...
12 October 2015 4,532 5 View
I wish to perform zymography for MMP-2 and MMP-9. I am working on cardiac remodeling and I have cardiac tissues as my sample to work on. I am confused if I shall extract protein from my tissue or...
05 October 2015 4,492 7 View
