New pages

5 June 2025

• 14:2714:27, 5 June 2025 Protein expression and purification (hist | edit) [7,148 bytes] ESO wikiadmin (talk | contribs) (Created page with "== '''Expression of  HisSUMO-hPOPX2 (SAK) – Part 1/2''' == -      '''''Bacteria Sample'':''' #570 BL21 DE3 pRos. pET24a His-SUMO_hPopx2_fulllength -      One day prior, aprox. 15:00 : inoculate 50 mL of antibiotics containing media with 3 colonies and incubate o/n at 37°C -      7 mL each of overnight culture were used to inoculate 3 x 333 mL of antibiotics (Kan, cam) containing LB-medium in a 1 L Erlenmeyer flask until OD = 0.7 -      When OD is reach...") Tag: Visual edit
• 14:1714:17, 5 June 2025 Testexpression in Pichia pastoris (hist | edit) [1,506 bytes] ESO wikiadmin (talk | contribs) (Created page with " == Procedure == * Inoculate 5 mL YPD medium with one '''big''' colony ** in 50 ml falcon tube (secure lid with tape, don’t tighten lid) * Grow o. n. 30 °C, 200 rpm * Centrifuge at 2000 g, 5 min * Resuspend the complete pellet in 5 mL BMMY medium * Measure the OD ** Use a 1:100 dilution for measurement * Start growth ** For starters you don’t need to dilute your suspension ** For optimization purposes you may want to adjust your starting OD *** Recommended is a sta...") Tag: Visual edit
• 14:0714:07, 5 June 2025 Transformation of Pichia pastoris (hist | edit) [1,499 bytes] ESO wikiadmin (talk | contribs) (Created page with " == Procedure == * Linearize 20 µg of plasmid of interest with EcoRV * Purify via EtOH precipitation: ** Adjust sample volume to 50 µL (MilliQ) ** Add 5 µL sodium acetate, vortex ** Add 125 µL ice cold EtOH abs.; Incubate on ice for 15 min ** Centrifuge 30 min, 14’000 rpm, 4 °C ** Discard the supernatant ** Wash the pellet with 500 µL colt EtOH 70 % ** Centrifuge 15 min, 14’000 rpm, 4 °C ** Remove the supernatant, dry pellet at RT ** Dissolve in 15-25 µL Mil...") Tag: Visual edit
• 14:0414:04, 5 June 2025 Generation of competent Pichia pastoris (hist | edit) [849 bytes] ESO wikiadmin (talk | contribs) (Created page with "{{Protokoll-Mix|Inhalt=Wildtype Pichia Pastoris yJC100, grown on YPD agar plate *YPD medium (yeast extract (10 g/L), peptone (20 g/L), glucose (20 g/L)) *Transformation buffer (Tris (10 mM) pH 7,5, Sorbitol (0,6 M), LiAc (100mM), DTT (10 mM)) *Sorbitol (1M) *Inoculation loop *Erlenmeyer flask 500 mL *Spectrometer for OD measurement *50 mL falcons}} ===== Procedure ===== -       Inoculate 5 mL of YPD medium with one big colony -       Grow 8 h at 30 °C, 200 rpm...") Tag: Visual edit
• 13:5613:56, 5 June 2025 Purification of GST-fusion proteins (hist | edit) [2,329 bytes] ESO wikiadmin (talk | contribs) (Created page with "== Procedure == The frozen bacteria pellets are thawed in 5 ml T-buffer on ice and resuspended. Afterwards the samples are incubated with 1 ml lysozyme for 30 min. The cells are broken through the addition of 20 ml lysis buffer and the treatment of ultrasonic sound (impulse 50%, 20sec, 3-5x) on ice water. 300 µl sepharose-solution is added to the suspension and incubated for 10 min with head-over-head rotation. Afterwards the lysate is centrifuged for 20 min at 16000 r...") Tag: Visual edit
• 13:5313:53, 5 June 2025 Expression of GST-fusion proteins (hist | edit) [2,003 bytes] ESO wikiadmin (talk | contribs) (Created page with "{{Protokoll-Mix|Inhalt=* *LB-Medium (10g Bacto-Trypton, 5g yeast extract, 5g NaCl, pH 7.0, ad 1l A. bidest) *Ampicillin (100 µg/ml) *IPTG (100 mM Isopropylthiogalactosid) *2xSDS sample buffer 125mM Tris-HCl (pH 6.8), 10% (v/v)-β- *Mercaptoethanol, 5% (w/v) SDS, 0.1% (w/v) Bromphenolblau, 20% (v/v) glycerin *T-buffer 100mM Tris (pH 8.0), 5 mM EDTA *GST-antibody}} == Procedure == === '''Test expression:''' === After cloning, the constructs of the desired GST-fusion pr...") Tag: Visual edit
• 13:5013:50, 5 June 2025 Bacterial Pulldown with soluble receptors (hist | edit) [1,616 bytes] ESO wikiadmin (talk | contribs) (Created page with "{{Protokoll-Mix|titel=Materials|Inhalt=PBS (1x) – 24g NaCl, 0.6g KCl, 3.45g Na2HPO4*7H2O, 0.6g KH2PO4, pH 7.4, ad 1l A.bidest<br>Cell culture supernatants (e.g. OptiMEM) harvested from 293cells transiently transfected with constructs encoding soluble CEACAM- ectodomains fused to GFP<br>Polyclonal rabbit anti-GFP antibody (AG Hauck)<br>2xSDS sample buffer 125mM Tris-HCl (pH 6.8), 10% (v/v) β-Mercaptoethanol, 5% (w/v) SDS, 0.1% (w/v) Bromphenolblau, 20% (v/v) Glycerin}}...") Tag: Visual edit
• 13:4813:48, 5 June 2025 Opsonizing bacteria with Fc fusion proteins (hist | edit) [599 bytes] ESO wikiadmin (talk | contribs) (Created page with "Bacteria are stained with CFSE and resuspended in PBS. 0.3 bacteria (OD 550) are incubated with either 0.05 mg of purified fusion proteins of 1ml of the unpurified fusion protein for 1 h under rotation at 4°C. As a positive control bacteria are incubated with 500 µl of anti-gonococcal sera (heat-inactivated for 1 h at 56°C) and 500 µl PBS. As a negative control bacteria are incubated with 1 ml of PBS. After the incubation the bacteria are washed and resuspended in 1...") Tag: Visual edit