ESO wikiadmin at 12:53, 5 June 2025

2025-06-05T12:53:59Z

← Older revision		Revision as of 14:53, 5 June 2025
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	{{Protokoll-Mix\|Inhalt=*		{{Protokoll-Mix\|Inhalt=<br>
	*LB-Medium (10g Bacto-Trypton, 5g yeast extract, 5g NaCl, pH 7.0,		*LB-Medium (10g Bacto-Trypton, 5g yeast extract, 5g NaCl, pH 7.0,
	ad 1l A. bidest)		ad 1l A. bidest)

ESO wikiadmin: 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..."

2025-06-05T12:53:43Z

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..."

New page

{{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 protein are transformed in ''E.coli'' BL21. Eight bacterial clones are selected for overexpression analysis. For the overexpression, bacterial clones are incubated in 5 ml antibiotica containing LB- medium for

6 h at 30°C and 220 rpm. After 6 h, 1ml of the culture is centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 µl 2xSDS sample buffer. The remaining culture is induced with 0.1 mM IPTG and incubated overnight at 30°C and 220 rpm. At the next day

0.5 ml of each sample is centrifuged for 1 min at 13000 rpm, the pellet is resuspended in 100 µl 2xSDS sample buffer and heated for 10 min at 90°C. The expression oft he protein is analysed by SDS-Page and Coomassie-staining.

=== Expression for purification: ===
One bacterial clone is incubated in 30 ml antibiotica containing LB-medium overnight at 30°C and 220 rpm. After 12-18h, 300 ml antibiotic containing LB-medium is inoculated with OD<sub>600</sub>=0,2 of the overnight-culture and incubated at 30°C and 220 rpm. At OD<sub>600</sub> = 0.6-0.8 1 ml is removed (sample before induction) and the remaining culture is induced with 0.5 mM IPTG for 3 h at 30°C and 220 rpm. Afterwards 0.5 ml is removed (sample after incuction), centrifuged for 1 min at 13000 rpm and the pellet is resuspended in 100 µl 2xSDS sample buffer and heated for 10 min at 90°C. The expression oft the protein is analysed by SDS-Page and Coomassie-staining. The remaining cells are centrifuged for 15 min at 4700 rpm, washed with T-buffer and stored at -80°C.

Expression of GST-fusion proteins - Revision history

ESO wikiadmin at 12:53, 5 June 2025