Can you help me on isolating biotinylated proteins from cell culture and detecting them in WB?

Hamdan Hamdan @Hamdan_Hamdan3

05 May 2014 1 6K Report

Mark Livingstone

The BioID paper has some detailed protocols: http://jcb.rupress.org/content/196/6/801.long

Some smaller companies supply nice magnetic streptavidin beads:

http://www.polysciences.com/Catalog/Department/Product/98/categoryid--593/productid--2575/

http://www.nanocs.net/Streptavidin-magnetic-nanoparticles-25nm-1.htm

Badges
Science method

More Hamdan Hamdan's questions See All

Does anyone have any recommendations or suggestions for neuronal specific crd line that is highly efficient and active?

The neuronal specific crd line.

07 August 2014 8,488 4 View

Does anyone know what might be causing the smeared bands on my western blot?

I got these smeared bands quite often lately. We typically run the gel at 140V with a 10-12% gel and do a wet transfer at 220 mA for 1.5 hr in cold room. We also noticed some dirty spots/dots (see...

10 August 2024 7,480 3 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

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

How to isolate lymphocytes from mouse spleen?

I have tried several times to isolate lymphocytes from mouse spleen, but all attempts have been unsuccessful. I tried most available protocols. I used different dissociation media (HBSS with Ca...

04 August 2024 9,913 7 View

What Are the Best Alternatives to the MTT Assay for Assessing Cell Toxicity of Colored Herbal Extracts?

I am currently investigating the cytotoxicity of a series of herbal extracts, and like many studies, I have been using the MTT assay to evaluate cell viability. However, I am encountering a...

31 July 2024 193 4 View

Assistance with MTT Assay Data ?

I did an MTT assay with 2 drugs (Piroxicam and Sulfameter) when I got my results for the 12,24,48,72 hours of incubation with my drug at the following concentrations [ug/ml]...

26 July 2024 7,511 3 View

Can I use sonicator only to lyse the exosomes and measure their protein contents using BCA assay kit?

Hello, I want to ask regarding exosome protein quantification method. Is it necessary to lyse exosomes before measuring their protein contents using BCA assay kit? Or can I just straight away...

23 July 2024 4,603 4 View

How do I avoid HEK293 cell lifting during biotinylation?

I’m doing cell-surface biotinylation with HEK293 cells and after all the washing and biotin incubation steps (washing with PBS ph8, neutralizing unreacted biotin with 40mM Tris ph8) many of my...

23 July 2024 4,740 3 View

How do we pick data for determination of Validation Acceptance Criteria?

Hello, colleagues! There is commenting open for new upcoming edition of USP 1033. Validation target acceptance criteria is now different from what it used to be and it doesn't include Cpm....

23 July 2024 7,292 3 View

My question concerns MTT cell viability assay?

Despite not having cells in the media, I am getting purple color. I have tried many troubleshooting methods, varying media types, and even different MTTs from different companies to figure out the...

21 July 2024 9,914 1 View