Can I use blocking peptide as positive control in Western blot?

I'm just curious if blocking peptides can be used in this fashion? Does the conjugation of the blocking peptide with the primary antibody stop the immunoreactivity with say a flouro-tagged...

31 December 2015 5,556 5 View

Contaminated taqman probe?

i think my taqman primer / probe is contaminated. is there any way to decontaminate? e.g., uv light, etc.

08 September 2015 171 2 View

MDA-MB-231 advice?

I'm looking to do a time course realtime-glo vitality assay on MDA-MB-231 in a 96-well plate. I plan on doing 4 different seeding concentrations to assess linearity of signal, however there is...

05 June 2015 1,797 4 View

How can I perform SiRNA concentration scaling? by nM or pmol per num of cell / surface area?

31 December 2014 4,760 6 View

How can I explain great siRNA transfection efficiency but no knockdown with validated positive controls?

I'm performing reverse transfection on SH-SY5Y using Viromer Green, and I get wonderful transfection efficiency (90+%). See picture (RFP labeled siRNA fluorescence overlapped on phase contrast...

10 November 2014 7,460 7 View

Can anybody recommend how much Blasticidin should be used for selection of SH-SY5Y?

We are electroporating a pcDNA6.2 plasmid contain miR RNAi constructs into SH-SY5Y. I plan on selecting these cells with blasticidin, and the literature suggests closer to the 10ug/ml mark for...

09 October 2014 6,422 3 View

Is anyone familiar with electroporation?

I thoroughly pipette my cells prior to electroporating so they should form a monolayer when plated. Though every time I electroporate, they clump. I'm not entirely sure why. When I passage, they...

08 September 2014 6,324 3 View

Shsy5y immunostaine troubleshooting

cells plated

07 August 2014 2,161 0 View

What is a good number of SH-SY5Y to freeze down for long term storage between 1-12mo?

What is a good number of SH-SY5Y to freeze down for long term storage between 1-12mo? Ideally, I'd like to initially plate in something larger like a t75 or t150, though I'm not sure if it is...

06 July 2014 4,719 2 View

What is the best method to carry out SH-SY5Y differentiation and transfection?

How do people typically go about electroporating differentiated SH-SY5Y? I was thinking that you would have to electroporate them first then recover in media supplemented with retinoic acid. Would...

06 July 2014 1,714 1 View

Does anyone have a recommendation for yeast reporter gene plasmid/protocol?

Hi, could anyone recommend a plasmid and/or protocol for reporter gene assay in S. cerevisiae? I want to assess the effect of growth conditions on a transcription factor, so I want to clone it`s...

01 March 2021 210 1 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

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

Expression cloning by using cDNA,do I need to modify the cDNA first?

I am going to have a expression cloning of mammalian gene by using shuttle plasmid to transforming the E.coli However I don't know I should only inserting the Coding sequence ,or I can...

28 February 2021 5,440 3 View

How does an antibody have full intact antibody + half antibody?

Hello, I was just wondering how does a antibody have full intact antibody + half antibody? Is it because of the drug conjugation?

28 February 2021 1,577 1 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

PUC19 as a transfer plasmid for lentiviral delivery?

Hello, Is it possible to use pUC19 as a transfer vector to be packed in using the second generation viral particles packaging system( pMD2.G; psPAX2 plasmids)? As far as I understand it there is...

28 February 2021 4,868 2 View

Does RNAiMAX transfection reagent work in serum free medium?

I diluted siRNA and RNAiMAX in opti-MEM and added to the cells which they were in the growth medium. Is it a right way? or should I culture cells in the opti-MEM medium for a while and not in...

26 February 2021 10,041 3 View

Did anyone ever try using pLVX-TetOne Plasmid with a copGFP as a selection marker instead of Puromycin?

When using a lentiviral vector for inducible expression of genes using the Tet-On system. In the literature, I saw a widely used of pLVX-TetOne-Puro Plasmid, is there a reason why people prefer to...

25 February 2021 1,011 3 View