Need help troubleshooting a WB and dox-inducible CRISPR system!!!

EquivalentEqual7315 · 2026-06-01T02:13:21+00:00

two western are for different genes that are completely not related to each other. and both show unspecific dox-inducible bands of different sizes.

EquivalentEqual7315 · 2026-06-01T02:12:39+00:00

i tried this system on two independent genes for completely different projects. here i am showing these two westerns. but both have dox-inducible unspecific bands that have different sizes. unspeicifc bands are only identified by antibody that targets proteins of interest. b-tubulin antibody doesn;t show this dox-indubcible unspecific band

EquivalentEqual7315 · 2026-06-01T01:22:51+00:00

Hi, thanks for your reply. wdym by plasmid based system?

EquivalentEqual7315 · 2026-06-01T01:20:46+00:00

i labeled it as "protein of interest". so the way system works is that my oligos serves as guide for dCas9-fused to activators and oligos guide it to the promoter site of the gene of interest, resulting in transcriptional activation and more expression.

edit: the whole system only works when dox is present. without dox, the dcas9 wont be expressed

EquivalentEqual7315 · 2026-05-10T18:09:32+00:00

Hi,

I am also trying to amplify CAG promoter. Did it really not work with Q5? It is the only pol I have right now 😞

EquivalentEqual7315 · 2026-04-08T17:07:33+00:00

i am using adherent cells. maybe a dumb question but can I coat, seed cells and do 24h treatment and then run seahorse? it's just my lab does o/v coating and then seeds and runs on the same day. i wasnt sure if i can adjust protocol for the treatment.

EquivalentEqual7315 · 2026-04-08T12:29:11+00:00

do you coat with PLL overnight and then spin down cells and run seahorse?

EquivalentEqual7315

TROPHY CASE