How to measure cGMP ( using kit from R&D system) from the mouse isolated myocytes ( myocytes from the heart after enzyme digestion)?

19 August 2016 0 6K Report

I bought cGMP essay kits from R&D system to measure cGMP from mouse cardiac myocytes. First I digest the mouse heart with collagenase ( perfused the enzyme through coronary artery using Langendroff set up) and discard the RV and atrias, then I triturate the LV so that the LV tissue releases alot of isolated intact cardiomyotyes into the solution. Thus at the end, I will get alot of intact cells suspend in the cell solution (One mouse LV in 10 ml of cell solution). My question is that how much cell suspension I should use to make cell lysate for cGMP essay?

Badges
Science topic

More Mei Methawasin's questions See All

Does anyone notice the tumor regression impact of Hepa1-6 cells in HCC orthotopic mice model?

To establish an orthotopic HCC model, C57BL/6 mice (male, 6-8 weeks) were orthotopically implanted with 3x106 Hepa1-6 cells (Over-expressed with a human gene) in 20uL Matrigel into the left lobe...

18 July 2024 9,177 2 View

Western blot showed distorted band, reverse 'U' shape, low MW protein?

This is 12% SDS PAGE gel and the MW of this protein is around 20 kDa. Running: 80V 20min; 110V 2h30min Transfer: 100V 1h20min I wonder this might be due to the gel over-heated during the running?

04 January 2024 7,414 0 View

What causes the faint and smeared bands in agarose gel electrophoresis?

The cDNA was amplified by Q5 and PCR product was analyzed by agarose gel electrophoresis. Would cDNA template degradation cause such smeared or smiling bands?

13 December 2023 3,031 12 View

How can I detect RIBOFLAVIN inside the cells?

Hi everyone, I got a question about the methods/assays I can use to detect Riboflavin inside the cells. Also after treatment with RF ? Fluorescence spectroscopy, HPLC, ELISA, seahorse??? If...

02 December 2023 3,001 0 View

Will endogenous and over-expressed protein level show different band size in western blot?

Pull down assay showed a discrepancy of band size between endogenous and over-expressed stable clones. Left: Endogenous Right: OE

30 October 2023 643 6 View

Why did DNA agarose gel electrophoresis show smearing band?

I used KAPA hifi pcr kit to linearize the plasmid for in-fusion cloning. Following the protocol, the DNA template amount was 5 ng and I already diluted it 100x. Annealing temperature: 66 69. The...

29 May 2023 5,232 5 View

As a beginner, how to use python to analyze RNA-seq data?

Hi, dear friends online. I am a fresh person in bioinformatics with little understanding. but I need to learn to analyze RNA-sequencing data. I think python is simple, I want to use it to...

05 April 2023 2,626 3 View

What is a good standardization approach when only part of a dataset is used in MLM?

Hello everybody, I'm using cluster-mean standardized repeated measures data (intensive longitudinal data) in multilevel models. However, I only used part of the data in the models (e.g., the...

10 January 2023 4,439 4 View

What is the difference between Lipofectamine 2000 and Lipofectamine 3000 for stable transfection of Lentivirus?

What is the difference between Lipofectamine 2000 and Lipofectamine 3000 for stable transfection of Lentivirus? I was told that Lipofectamine 3000 is more toxic to the cells with higher...

12 December 2022 7,036 4 View

Can I use error-prone PCR to do random mutagenesis in 5 kb fragments ?

I want to do random mutagenesis in 5 kb fragments. I want to use the product to do directed evolution. I used two error-prone PCR kits (Aligent and Takara) and can't amplify fragments which size...

29 October 2022 3,154 4 View

What are the long-term impacts of incarceration on youths' developing brain?

I want to explore the long-term effects of incarceration on a youth's developing brain. I also want to explore research that looks critically at incarceration and punitive measures as the primary...

12 August 2024 862 0 View

Why Do TDS and EC Increase with Larger Wastewater Volumes, While BOD and COD Decrease?

I have carried out MFC experiments on three different volumes, 50, 500 and 1000 mL of wastewater. Results after MFC treatment shows that TDS and EC are more in larger volumes of water i.e. TDS and...

09 August 2024 9,621 0 View

Could it be a cell culture contamination?

Hello everyone! I observed in my culture (htert-RPE1 cells) an orange- red particle at the bottom of the dish. It is visible to the naked eye as a very very small red dot. Could it be a...

09 August 2024 2,824 3 View

Does Anyone have expertise in in vitro transcription and RNA pull down assay?

I am currently working on LncRNA; to know the lncRNA-protein interactions I want to do RNA pull down assay, so I need to design primers with T7 promoter. I need assistance in this regard.

07 August 2024 6,622 1 View

Is there any way to quantify bacterial and fungal cells in their mixed culture?

I am working in fungal fermentation of soybean meal and there is bacterial growth in them at times. I am trying to quantify fungal cell counts and bacterial cells; but I haven't been able to do at...

07 August 2024 7,535 4 View

How to normalize and take the significance of the MTT OD values with 3 replicates for the same cell-line?

Hi, I have a question about normalizing the MTT OD values for doing the statistical analysis. So, if we have 3 different plates and we call them 3 different replicates, so, first we would...

07 August 2024 8,106 4 View

How to improve plaques in Double Layer Agar Assay?

Hi all! My thesis groupmates and I are working on purifying an E. coli bacteriophage. The phage was handed down to us by the previous thesis group, but they only managed to purify it to about...

06 August 2024 5,801 4 View

Why activated CAR-Jurkat cell could not kill targets?

Previously when I co-coluture anti-CD19(FMC63) CAR-Jurkat with Raji with E:T=5:1, Jurkat can eliminate Raji in 24h. However, when I test another CAR construct, although I can dectect totally CD69...

06 August 2024 641 2 View

Are there instances where molecules with larger molecular weights exhibit greater mobility than those with smaller molecular weights?

Hi, I know that low molecular weight (MW) molecules generally tend to have higher mobility, while high molecular weight molecules tend to have lower mobility. However, in my experimental...

06 August 2024 1,495 2 View

How much total RNA concentration to be extracted from sorted plasma cells from bone marrow of C57BL/6 mice for RT-PCR ?

i have sorted anti-NP specific plasma cells from bone marrow of C57BL/6 mice at certain times after immunization with variable counts and isolated total RNA using TRIZOL method for RT-PCR using...

05 August 2024 8,835 1 View