Gowthami Mahendran

10 Questions 15 Answers 0 Followers

Questions related from Gowthami Mahendran

I am facing problems of getting rid of the IgG bands in IP blots. How to avoid these IgG bands in the blots to prevent it overlapping?

I am working on pulling down the protein of interest from human cell lysates. My band of interest is in the 40 kDa area, and I am having problems detecting them due to the IgG bands. I see people...

18 February 2025 9,856 4 View

What is considered a biological replicate when you are doing an ELISA plate measurement with different cell types?

If cells obtained from different passages, with different plating but harvested on the same day are used in a single plate to take an ELISA measurement (with the given standards and treated...

18 June 2024 6,769 1 View

Which phosphor antibodies to choose to study the hypo and hyperphosphorylation state of the SRSF1 protein?

I am trying to understand the different phosphorylation states of SRSF1 protein in the cytoplasm vs nucleus and looking for specific antibodies for the phosphor state. As SR proteins include...

12 February 2024 7,281 3 View

Is it necessary to change media after transfecting cells with Lipofectamine3000?

I transfected HCt116 cells with GFP using Lipo3000. I didn't change media after transfection as the manufacturer guidelines say not necessary. However, I noticed a change in the cell morphology...

29 April 2023 6,109 6 View

Is Native RNA immunoprecipitation performed without cross linking?

I am trying to study the RNA-protein interactome of a specific protein using IP studies. I need to use native IP for my purpose and wanted to know if native RIP is without cross-linking study and...

02 February 2023 948 0 View

Is loading equal volume of protein in SDS PAGE correct?

I am comparing the protein expression levels in subcellular fractionations (Cytoplasm and nuclear). If I load an equal volume of both extracts (15 ul each to individual wells), is this still valid...

27 January 2023 6,298 3 View

What is the right media for HCT116?

Is DMEM or DMEM/F-12K the right media for culturing HCT116 cells apart from McCoys media? Did anyone use the gibco one? I see a lot of dead cells when using DMEM. It would be helpful if someone...

21 June 2022 1,106 3 View

How is FPKM calculated from raw gene count file?

I am working with RNAseq and qRT-PCR validation results to see a correlation. I am comparing a diseased group to a healthy group of cells. I have 4 biological replicates in each category. However,...

04 June 2022 8,036 5 View

BN-PAGE gradient gels?

I have tried using BN-PAGE for mitochondrial complexes isolation. I have followed several protocols (Nature protocols-2006, JOVE-2017). But I experienced a problem with running the gel. The dye...

27 June 2020 5,640 6 View

What is the purpose of adding detergent during sample prep for the BN PAGE and how to find the volume of it for mitochondrial complex separation?

I am working with BN PAGE for the first time and I am not sure about the amounts of detergent (digitonin) that should be added. Also, I am looking for methods to find the concentration of the...

17 March 2020 2,288 9 View