Purification of GST-fusion proteins: Difference between revisions

Revision as of 13:59, 5 June 2025

Materials

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

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.

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   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  Aprotinin [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.

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Purification of GST-fusion proteins: Difference between revisions

Revision as of 13:59, 5 June 2025

Procedure

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