Q&A Jun 10, 2026

tRNA charging protocolDay 0: Cell PlatingPlate 300,000 cells per well of a 6-well plate per condition (I do 4 conditions in singlicates at a time).Day 1: Cell treatment and harvesting Treat cells with treatments of your choice;Put cells on ice and wash with cold PBS;Add 1 mL TRIzol on ice, lyse for 5 min;Add lysates to eppendorf tubes with 0.2 mL chloroform, shake vigorously 15 sec and let sit on ice for 10 min;Spin down for 15 min at 18,600g at 4C;Transfer 370 uL of top fraction into new tubes ...

Q&A Jun 9, 2026

 Day 1 Cell seeding  Seed U2OS cells in 10 cm dishSeed 1 Mio cells per dishSeed 3 biological replicates per condition Day 2 Cell synchronization and treatment of cells  NOTE: Timing are stated as an example for handling 3 biological replicates 16.01.25 Block 1st round (15:00, 15:20, 15:40) Remove culturing mediumAdd fresh medium supplemented with thymidine to a final concentration of 2 mM (1:50)Culture cells in a tissue culture incubator at 37 °C for 18 h 17.01.24 Release (9:00, 9:20, 9:40) Remo...

Q&A Jun 9, 2026

Starve Cal51 cells by incubating in DMEM-0 (0% serum) overnight (more than 20 hr).Trypsinize the starved Cal51 cells and resuspend cells with DMEM-10 (10% serum). The serum used here is to neutralize Trypsin to minimize the cytotoxicity.Count the cell number using a hemocytometer under microscope. Pellet the cells to the bottom of a 1.5-ml microcentrifuge tube by centrifugation (800 g, RT, 2 min).Aspirate the media and resuspend cells with an appropriate amount of DMEM-0. Pipette 30 times to ful...

Q&A Jun 1, 2026

One 10 cm dish of wild type HEK293T cells were grown to ~80% confluency for each replicate of each experiment. Cells were scraped from the dish and pipetted with media into a 15 ml conical vial.The cells were pelleted by centrifugation at 300 xG for 2 minutes and media was aspirated and discareded. The cell pellet was then resuspended  2 ml of ice cold PBS, centrifuged at 300 xG for 2 minutes and PBS was aspirated and discarded. This PBS wash was repeated for a total of 2 washes. The cell pellet...

Q&A May 28, 2026

Screening of protein kinase candidates in silico and in vitro  I. In silico screen:The recognition sequence for phosphorylation varies from kinase to kinase and contains a variable number of amino acids. These can be located both before and after the phosphorylated amino acid. The type of amino acids required for recognition also varies greatly. The in silico analysis was used to generate an overview of kinases predicted to phosphorylate Su(H) at S269. To this end we applied the GPS 3.0 software...

Q&A May 26, 2026

In Vitro mTOR - Claspin Binding Assay Protocol AbstractProtein-protein interactions can be assessed by many procedures. Among them, immunoprecipitation with a specific antibody is one of the most commonly used methods. Here, we describe a method to investigate the interactions using purified proteins.This protocol was designed to observe the interaction between Claspin and mTOR proteins using immunoprecipitation but should be applicable to the interactions of any proteins.Keywords: protein-prote...

Q&A May 15, 2026

β-Glucuronidase Activities of FecesTo determine β-glucuronidase activities, we mixed 20 μL of the fecal suspension with 180 μL of the reaction mixture containing 1 mM p-nitrophenyl-β-D-glucuronide (FUJIFILM Wako), 50 mM HEPES-HCl (pH 7.4), and 37.2 mM 2-mercaptoethanol (24). We incubated the mixture at 37°C and measured its optical density (OD) at 405 nm, every 5 min between 10- and 30-min incubation times. We calculated the average OD change per min. Preparation of Fecal suspensionWe used fresh...

Q&A May 11, 2026

Puromycin Assay for detection of newly synthesized proteins  Puromycin Dihydrochloride powder (Sigma-CAS 58-58-2) is dissolved in milli-Q water and stored at -20 °C in 1 ml aliquots (Stock concentration - 25 mg/ml).Treat cell lines with puromycin (final concentration – 10 micrograms/ml) in media for 20 min. Wash cells with 1X PBS and collect them for lysis. Have “no puro treated” cells as a negative control for the assay (add same volume of water as vehicle).Run Western blot using the lysates (l...

Q&A May 11, 2026

MINFLUX data postprocessing protocol Author: Dr. Charlotte Kaplan             Super-resolution microscopy specialist             CellNetworks Core Technology Platform             Heidelberg University, Bioquant             Im Neuenheimer Feld 267             Heidelberg, Germany             Contact: charlotte.kaplan@bioquant.uni-heidelberg.de Introduction:MINFLUX data analysis procedures and open source tools have develop extensively sincethe publication of the first commercial MINFLUX systems (S...

Q&A May 7, 2026

F/G Actin separation protocol  It is important that ALL buffers are made fresh and are ice-cold prior to use. Lysis buffer recipe (for G-actin supernatant)10mM K2PO4100mM NaF50mM KCl2mM MgCl21mM EGTA0.2mM DTT0.5% Triton-X 1001mM sucrose pH 7.0 Second lysis buffer (for F-actin supernatant)1.5mM guanidine hydrochloride1mM sodium acetate1mM CaCl21mM ATP20mM Tris-HClpH 7.5 ProtocolPrepare both lysis buffers in diH2O and place on ice. Lyse cells or homogenates with sufficient volume of G-actin supern...