Purification of GST-fusion proteins - Revision history

ESO wikiadmin at 13:03, 5 June 2025

2025-06-05T13:03:29Z

← Older revision		Revision as of 15:03, 5 June 2025
Line 27:		Line 27:

	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 rpm and 4°C. For purification, 1-2 ml 50% glutathione-sepharose solution (GST- Fast4Flow in T-buffer) is added to the lysate, which was washed three times with T- buffer. The sample is then incubated for 2-4 h at 4°C with head-over-head rotation. Afterwards the pellet is washed three times with 1x PBS by centrifugation at 2000 g. The supernatants (wash1-wash3) are collected each time and stored at 4°C. The success of the purification is analysed by SDS-PAGE and Coomassie-staining. The isolated, purified GST-fusion protein coupled to gluthathione-sepharose can now be used for the GST-pulldown. The remaining sample is eluted by the addition of glutathione and is stored at -80°C after dialysis.		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 rpm and 4°C. For purification, 1-2 ml 50% glutathione-sepharose solution (GST- Fast4Flow in T-buffer) is added to the lysate, which was washed three times with T- buffer. The sample is then incubated for 2-4 h at 4°C with head-over-head rotation. Afterwards the pellet is washed three times with 1x PBS by centrifugation at 2000 g. The supernatants (wash1-wash3) are collected each time and stored at 4°C. The success of the purification is analysed by SDS-PAGE and Coomassie-staining. The isolated, purified GST-fusion protein coupled to gluthathione-sepharose can now be used for the GST-pulldown. The remaining sample is eluted by the addition of glutathione and is stored at -80°C after dialysis.

	~~<!DOCTYPE html>~~
	~~<html lang="de">~~
	~~<head>~~
	~~<meta charset="UTF-8">~~
	~~<meta name="viewport" content="width=device-width, initial-scale=1.0">~~
	~~<title>Laboratory Recipes</title>~~
	~~<style>~~
	~~body {~~
	~~font-family: monospace;~~
	~~line-height: 1.4;~~
	~~margin: 20px;~~
	}
	~~.recipe-section {~~
	~~margin-bottom: 15px;~~
	}
	~~.ingredient-name {~~
	~~display: inline-block;~~
	~~width: 200px;~~
	~~vertical-align: top;~~
	}
	~~.concentration {~~
	~~display: inline-block;~~
	~~width: 120px;~~
	}
	~~.arrow {~~
	~~margin: 0 10px;~~
	}
	~~.final-conc {~~
	~~display: inline-block;~~
	}
	~~.indent {~~
	~~margin-left: 200px;~~
	}
	~~</style>~~
	~~</head>~~
	~~<body>~~

	~~<div class="recipe-section">~~
	~~<span class="ingredient-name"><strong>LB-Medium</strong></span>~~
	~~<span>10g Bacto-Trypton, 5g yeast extract, 5g NaCl, pH 7.0,<br>~~
	~~<span class="indent">ad 1l A.bidest</span></span>~~
	~~</div>~~

	~~<div class="recipe-section">~~
	~~<span class="ingredient-name"><strong>Ampicillin</strong></span>~~
	~~<span>100μg/ml</span>~~
	~~</div>~~

	~~<div class="recipe-section">~~
	~~<span class="ingredient-name"><strong>IPTG</strong></span>~~
	~~<span>100mM Isopropylthiogalactosid</span>~~
	~~</div>~~

	~~<div class="recipe-section">~~
	~~<span class="ingredient-name"><strong>T-buffer</strong></span>~~
	~~<span>100mM Tris (pH 8.0), 5mM EDTA</span>~~
	~~</div>~~

	~~<div class="recipe-section">~~
	~~<span class="ingredient-name"><strong>Lysis buffer (200 ml):</strong></span>~~
	~~<div class="indent">~~
	~~10  ml  Tris pH 8.0 [1M]<span class="arrow">→</span>50 mM<br>~~
	~~4   ml  EDTA [0.5 M]<span class="arrow">→</span>10 mM<br>~~
	~~30  ml  NaCl [1M]<span class="arrow">→</span>150 mM<br>~~
	~~40  ml  Glycerol [50 %]<span class="arrow">→</span>10 %<br>~~
	~~0.5 ml  Triton [10 %]<span class="arrow">→</span>0.025 %<br>~~
	~~<strong>add freshly à 20 ml:</strong><br>~~
	~~50  μl  DTT [1M]<span class="arrow">→</span>2.5 mM<br>~~
	~~20  μl  PMSF [10 mg/ml]<br>~~
	~~20  μl  Leupeptin [10 mg/ml]<br>~~
	~~20  μl  Aprotinin [10 mg/ml]<br>~~
	~~20  μl  Pefablock [10 mg/ml]~~
	~~</div>~~
	~~</div>~~

	~~<div class="recipe-section">~~
	~~<span class="ingredient-name"><strong>Lysozyme</strong></span>~~
	~~<span>1 mg/ml</span>~~
	~~</div>~~

	~~<div class="recipe-section">~~
	~~<span class="ingredient-name"><strong>Sepharose- solution,</strong></span><br>~~
	~~<span class="ingredient-name"><strong>Glutathione-sepharose-solution</strong></span>~~
	~~</div>~~

	~~<div class="recipe-section">~~
	~~<span class="ingredient-name"><strong>PBS (1x)</strong></span>~~
	~~<span>24g NaCl, 0.6g KCl, 3.45g Na₂HPO₄ x 7 H₂O, 0.6 g<br>~~
	~~<span class="indent">KH₂PO₄, pH 7.4, ad 1l mit A.bidest.</span></span>~~
	~~</div>~~

	~~</body>~~
	~~</html>~~

ESO wikiadmin at 13:03, 5 June 2025

2025-06-05T13:03:16Z

← Older revision		Revision as of 15:03, 5 June 2025
Line 1:		Line 1:
	{{Protokoll-Mix\|titel=Materials\|Inhalt=LB-Medium 10g Bacto-Trypton, 5g yeast extract, 5g NaCl, pH 7.0, ad 1l A.bidest		{{Protokoll-Mix\|titel=Materials\|Inhalt=<pre>
	Ampicillin ~~100µg~~/ml		LB-Medium 10g Bacto-Trypton, 5g yeast extract, 5g NaCl, pH 7.0,
	IPTG 100mM Isopropylthiogalactosid		ad 1l A.bidest
	T-buffer 100mM Tris (pH 8.0), 5mM EDTA		Ampicillin 100μg/ml
	Lysis buffer (200 ml): 10 ml Tris pH 8.0 [1M]  50 mM		IPTG 100mM Isopropylthiogalactosid
	4 ml EDTA [0.5 M]  10 mM		T-buffer 100mM Tris (pH 8.0), 5mM EDTA
	30 ml NaCl [1M]  150 mM		Lysis buffer (200 ml): 10 ml Tris pH 8.0 [1M] → 50 mM
	40 ml Glycerol [50 %]  10 %		4 ml EDTA [0.5 M] → 10 mM
	0.5 ml Triton [10 %]  0.025 %		30 ml NaCl [1M] → 150 mM
	add freshly à 20 ml:		40 ml Glycerol [50 %] → 10 %
	50 µl DTT [1M]  2.5 mM		0.5 ml Triton [10 %] → 0.025 %
	20 µl PMSF [10 mg/ml]		add freshly à 20 ml:
	20 µl Leupeptin [10 mg/ml]		50 μl DTT [1M] → 2.5 mM
	20 ~~µl Aprotenin~~ [10 mg/ml]		20 μl PMSF [10 mg/ml]
	20 µl Pefablock [10 mg/ml]		20 μl Leupeptin [10 mg/ml]
	Lysozyme 1 mg/ml		20 μl Aprotinin [10 mg/ml]
	Sepharose- solution. Glutathione-sepharose-solution		20 μl Pefablock [10 mg/ml]
	PBS (1x) 24g NaCl, 0.6g KCl, 3.45g ~~Na2HPO4~~ x 7 ~~H2O~~, 0.6 g		Lysozyme 1 mg/ml
	~~KH2PO4~~, pH 7.4, ad 1l mit A.bidest.}}		Sepharose- solution,
			Glutathione-sepharose-solution
			PBS (1x) 24g NaCl, 0.6g KCl, 3.45g Na₂HPO₄ x 7 H₂O, 0.6 g
			KH₂PO₄, pH 7.4, ad 1l mit A.bidest.
			</pre>}}

	== Procedure ==		== Procedure ==

ESO wikiadmin at 12:59, 5 June 2025

2025-06-05T12:59:43Z

← Older revision		Revision as of 14:59, 5 June 2025
Line 23:		Line 23:

	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 rpm and 4°C. For purification, 1-2 ml 50% glutathione-sepharose solution (GST- Fast4Flow in T-buffer) is added to the lysate, which was washed three times with T- buffer. The sample is then incubated for 2-4 h at 4°C with head-over-head rotation. Afterwards the pellet is washed three times with 1x PBS by centrifugation at 2000 g. The supernatants (wash1-wash3) are collected each time and stored at 4°C. The success of the purification is analysed by SDS-PAGE and Coomassie-staining. The isolated, purified GST-fusion protein coupled to gluthathione-sepharose can now be used for the GST-pulldown. The remaining sample is eluted by the addition of glutathione and is stored at -80°C after dialysis.		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 rpm and 4°C. For purification, 1-2 ml 50% glutathione-sepharose solution (GST- Fast4Flow in T-buffer) is added to the lysate, which was washed three times with T- buffer. The sample is then incubated for 2-4 h at 4°C with head-over-head rotation. Afterwards the pellet is washed three times with 1x PBS by centrifugation at 2000 g. The supernatants (wash1-wash3) are collected each time and stored at 4°C. The success of the purification is analysed by SDS-PAGE and Coomassie-staining. The isolated, purified GST-fusion protein coupled to gluthathione-sepharose can now be used for the GST-pulldown. The remaining sample is eluted by the addition of glutathione and is stored at -80°C after dialysis.

			<!DOCTYPE html>
			<html lang="de">
			<head>
			<meta charset="UTF-8">
			<meta name="viewport" content="width=device-width, initial-scale=1.0">
			<title>Laboratory Recipes</title>
			<style>
			body {
			font-family: monospace;
			line-height: 1.4;
			margin: 20px;
			}
			.recipe-section {
			margin-bottom: 15px;
			}
			.ingredient-name {
			display: inline-block;
			width: 200px;
			vertical-align: top;
			}
			.concentration {
			display: inline-block;
			width: 120px;
			}
			.arrow {
			margin: 0 10px;
			}
			.final-conc {
			display: inline-block;
			}
			.indent {
			margin-left: 200px;
			}
			</style>
			</head>
			<body>

			<div class="recipe-section">
			<span class="ingredient-name"><strong>LB-Medium</strong></span>
			<span>10g Bacto-Trypton, 5g yeast extract, 5g NaCl, pH 7.0,<br>
			<span class="indent">ad 1l A.bidest</span></span>
			</div>

			<div class="recipe-section">
			<span class="ingredient-name"><strong>Ampicillin</strong></span>
			<span>100μg/ml</span>
			</div>

			<div class="recipe-section">
			<span class="ingredient-name"><strong>IPTG</strong></span>
			<span>100mM Isopropylthiogalactosid</span>
			</div>

			<div class="recipe-section">
			<span class="ingredient-name"><strong>T-buffer</strong></span>
			<span>100mM Tris (pH 8.0), 5mM EDTA</span>
			</div>

			<div class="recipe-section">
			<span class="ingredient-name"><strong>Lysis buffer (200 ml):</strong></span>
			<div class="indent">
			10  ml  Tris pH 8.0 [1M]<span class="arrow">→</span>50 mM<br>
			4   ml  EDTA [0.5 M]<span class="arrow">→</span>10 mM<br>
			30  ml  NaCl [1M]<span class="arrow">→</span>150 mM<br>
			40  ml  Glycerol [50 %]<span class="arrow">→</span>10 %<br>
			0.5 ml  Triton [10 %]<span class="arrow">→</span>0.025 %<br>
			<strong>add freshly à 20 ml:</strong><br>
			50  μl  DTT [1M]<span class="arrow">→</span>2.5 mM<br>
			20  μl  PMSF [10 mg/ml]<br>
			20  μl  Leupeptin [10 mg/ml]<br>
			20  μl  Aprotinin [10 mg/ml]<br>
			20  μl  Pefablock [10 mg/ml]
			</div>
			</div>

			<div class="recipe-section">
			<span class="ingredient-name"><strong>Lysozyme</strong></span>
			<span>1 mg/ml</span>
			</div>

			<div class="recipe-section">
			<span class="ingredient-name"><strong>Sepharose- solution,</strong></span><br>
			<span class="ingredient-name"><strong>Glutathione-sepharose-solution</strong></span>
			</div>

			<div class="recipe-section">
			<span class="ingredient-name"><strong>PBS (1x)</strong></span>
			<span>24g NaCl, 0.6g KCl, 3.45g Na₂HPO₄ x 7 H₂O, 0.6 g<br>
			<span class="indent">KH₂PO₄, pH 7.4, ad 1l mit A.bidest.</span></span>
			</div>

			</body>
			</html>

ESO wikiadmin at 12:59, 5 June 2025

2025-06-05T12:59:35Z

← Older revision		Revision as of 14:59, 5 June 2025
Line 1:		Line 1:
	{{Protokoll-Mix\|titel=Materials}}		{{Protokoll-Mix\|titel=Materials\|Inhalt=LB-Medium 10g Bacto-Trypton, 5g yeast extract, 5g NaCl, pH 7.0, ad 1l A.bidest
			Ampicillin 100µg/ml
			IPTG 100mM Isopropylthiogalactosid
			T-buffer 100mM Tris (pH 8.0), 5mM EDTA
			Lysis buffer (200 ml): 10 ml Tris pH 8.0 [1M]  50 mM
			4 ml EDTA [0.5 M]  10 mM
			30 ml NaCl [1M]  150 mM
			40 ml Glycerol [50 %]  10 %
			0.5 ml Triton [10 %]  0.025 %
			add freshly à 20 ml:
			50 µl DTT [1M]  2.5 mM
			20 µl PMSF [10 mg/ml]
			20 µl Leupeptin [10 mg/ml]
			20 µl Aprotenin [10 mg/ml]
			20 µl Pefablock [10 mg/ml]
			Lysozyme 1 mg/ml
			Sepharose- solution. Glutathione-sepharose-solution
			PBS (1x) 24g NaCl, 0.6g KCl, 3.45g Na2HPO4 x 7 H2O, 0.6 g
			KH2PO4, pH 7.4, ad 1l mit A.bidest.}}

	== Procedure ==		== Procedure ==

ESO wikiadmin at 12:56, 5 June 2025

2025-06-05T12:56:45Z

← Older revision		Revision as of 14:56, 5 June 2025
Line 1:		Line 1:
			{{Protokoll-Mix\|titel=Materials}}

	== Procedure ==		== 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.		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 rpm and 4°C. For purification, 1-2 ml 50% glutathione-sepharose solution (GST- Fast4Flow in T-buffer) is added to the lysate, which was washed three times with T- buffer. The sample is then incubated for 2-4 h at 4°C with head-over-head rotation. Afterwards the pellet is washed three times with 1x PBS by centrifugation at 2000 g. The supernatants (wash1-wash3) are collected each time and stored at 4°C. The success of the purification is analysed by SDS-PAGE and Coomassie-staining. The isolated, purified GST-fusion protein coupled to gluthathione-sepharose can now be used for the GST-pulldown. The remaining sample is eluted by the addition of glutathione and is stored at -80°C after dialysis.		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 rpm and 4°C. For purification, 1-2 ml 50% glutathione-sepharose solution (GST- Fast4Flow in T-buffer) is added to the lysate, which was washed three times with T- buffer. The sample is then incubated for 2-4 h at 4°C with head-over-head rotation. Afterwards the pellet is washed three times with 1x PBS by centrifugation at 2000 g. The supernatants (wash1-wash3) are collected each time and stored at 4°C. The success of the purification is analysed by SDS-PAGE and Coomassie-staining. The isolated, purified GST-fusion protein coupled to gluthathione-sepharose can now be used for the GST-pulldown. The remaining sample is eluted by the addition of glutathione and is stored at -80°C after dialysis.

ESO wikiadmin: 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..."

2025-06-05T12:56:19Z

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..."

New page

== 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 rpm and 4°C. For purification, 1-2 ml 50% glutathione-sepharose solution (GST- Fast4Flow in T-buffer) is added to the lysate, which was washed three times with T- buffer. The sample is then incubated for 2-4 h at 4°C with head-over-head rotation. Afterwards the pellet is washed three times with 1x PBS by centrifugation at 2000 g. The supernatants (wash1-wash3) are collected each time and stored at 4°C. The success of the purification is analysed by SDS-PAGE and Coomassie-staining. The isolated, purified GST-fusion protein coupled to gluthathione-sepharose can now be used for the GST-pulldown. The remaining sample is eluted by the addition of glutathione and is stored at -80°C after dialysis.