Knockdown efficiency reflected in PCR but not western blot?

Hi, I am doing siRNA of a particular gene of interest. however, after several attempts, western blot is not showing evidence of this knockdown, I have tried different antibodies and also longer...

02 March 2019 4,500 4 View

What is the best method for quantifying phalloidin staining?

I did a phalloidin staining for f-actin, Is it possible to quantify the f-actin stretches between two different staining and how?

03 April 2018 9,037 0 View

What is the best way to seed cells on acrylamide coated coverslips?

i am trying to seed cells on coverslip coated with gels (acrylamide based hydrogels) My main obstacle is my cells aren't settling down as expected so they can grow instead they keep floating, or...

10 November 2015 968 4 View

Any advice on Ethidium Bromide and Calcenin staining for cells seeded on coverslip?

I am trying to stain cells seeded on hydrogel coverslip with EtBr and Calcenin in 2D culture. Any suggestion on the best protocol to follow would be greatly appreciated.

10 November 2015 5,452 3 View

Why does my citrate buffer turn milky during immunohistochemistry?

For my IHC protocol, I am staining for beta-catenin in ovarian sections during antigen retrieval. My samples are heated in citrate buffer for 45 mins. I have also read that signs to show that your...

11 December 2014 6,213 4 View

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02 March 2021 3,060 3 View

Issues with Permeabilization during Flow Cytometry?

Hello Everyone. Currently I am working to characterize macrophages in the myocardium after ischemia-reperfusion injury in rats. Due to the low total cell number isolated from rat hearts I can...

01 March 2021 3,867 3 View

Which cell line should we use to perform biological dosimetry experiments?

We are preparing some experiments based on irradiating cells under different conditions in order to evaluate the effects in terms of DNA damage, genetic expression, etc. As our project is...

01 March 2021 3,355 3 View

Why might my GFP tag be dissociating from my plasmid after transfection?

I have a set of stably transfected cell lines all transfected with plasmids containing GFP tags on the C terminus. During a western blot using anti-GFP antibody, one of my plasmids has dissociated...

01 March 2021 9,310 4 View

Is it possible for two human neuronal cell lines to have different gene sequence for the same protein?

I am looking at the ATP1A2 (Sodium/Potassium ATPase alpha subunit 2) in two human neuronal cell lines. Expression levels of this protein seems to be almost equal when detected by one antibody....

01 March 2021 3,607 3 View

Can someone suggest a way to prepare slides to observe under confocal microscope for observing fluorescent tagged proteins??

I am growing the cells on coverslips and transfecting the fluorescent tagged protein directly on them. Then i want to observe these cells under confocal microscope. Currently i am just using...

01 March 2021 6,142 3 View

Is CD44 degraded in lysosome together with hyaluronan after CD44 mediated endocytosis of hyaluronic acid?

Also when RHAMM binds hyaluronic acid, they get internalized, will RHAMM also be degraded? Or both CD44 and RHAMM will be transferred back to the cell membrane? Asking for breast cancer cell line...

01 March 2021 8,169 2 View

Do you need the CMV enhancer before the CMV promotor for a proper function of CMV on the gene that follows?

I'm a student and I have to produce a cell line with a knockin for the NRF2 gene with GFP. I have to put a promotor in front of the GFP because the gene will be too far away from the promotor of...

28 February 2021 7,127 2 View

Problem with shRNA transfection. Why are only two plates getting contaminated instead of all?

I transfected my LNCaP-WT cells with 3 shRNA plus their NTC two weeks ago and split two puromycin selected cell plates on Friday last week(Feb 26). I checked for GFP in the cells, and they all...

28 February 2021 4,949 3 View

Interested to stain mice tissue with WGA 488 conjugate, anybody has a protocol to recommend?

Interested to stain mice brain tissue with WGA 488 conjugate, appreciate if anyone willing to offer me some guidance or suggestions! Thank you!

28 February 2021 3,951 2 View