Purification of GST Fusion Proteins: Difference between revisions

(2 intermediate revisions by the same user not shown)

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#Pellet beads (save sample of unbound protein) and wash 3 x 50 mL cold wash buffer being careful to not suck up beads

#Pour into disposable column (BioRad # 732-6008) to collect beads. Wash with another 10 mL of PBS

#Prepare elution buffer containing 50mM Glutathionie in PBS(0.615g in 40mL PBS), adjust pH to between 7 and 8.

#Elute with ~5 mL room temp. elution buffer, collecting 10 x ~0.5 mL fractions. Check fractions for protein with Bradford assay

*Elution buffer contains 50mM Glutathionie in PBS(0.615g in 40mL PBS), pH to between 7 and 8.

#Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X) or other desired buffer

#Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X)

#Measure protein concentration ([[Bradford Assay]] or [[Quantification by Absorbance at 280nm]]; concentrate if necessary and store at -20)

#Measure protein concentration (Bradford or ~~A280~~; concentrate if necessary and store at -20)

#Analyze uninduced and induced cells (0.5 mL culture resuspended in 100 uL 2X SDS), lysate (5 uL in 2X), unbound (5 uL in 2X) and purified protein (0.5-10 ug) by SDS-PAGE

#Analyze uninduced and induced cells (0.5 mL culture resuspended in 100 uL 2X SDS), lysate (5 uL in 2X), unbound (5 uL in 2X) and protein (0.5-10 ug) by SDS-PAGE

[[Category:Protein Purification]]

@@ Line 16: / Line 16: @@
 #Pellet beads (save sample of unbound protein) and wash 3 x 50 mL cold wash buffer being careful to not suck up beads
 #Pour into disposable column (BioRad # 732-6008) to collect beads.  Wash with another 10 mL of PBS
+#Prepare elution buffer containing 50mM Glutathionie in PBS(0.615g in 40mL PBS), adjust pH to between 7 and 8.
 #Elute with ~5 mL room temp. elution buffer, collecting 10 x ~0.5 mL fractions.  Check fractions for protein with Bradford assay
-*Elution buffer contains 50mM Glutathionie in PBS(0.615g in 40mL PBS), pH to between 7 and 8.
+#Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X) or other desired buffer
-#Combine fractions with protein and dialyse O/N into 200 mL PBS in 50% glycerol (will concentrate sample ~4X)
+#Measure protein concentration ([[Bradford Assay]] or [[Quantification by Absorbance at 280nm]]; concentrate if necessary and store at -20)
-#Measure protein concentration (Bradford or A280; concentrate if necessary and store at -20)
+#Analyze uninduced and induced cells (0.5 mL culture resuspended in 100 uL 2X SDS), lysate (5 uL in 2X), unbound (5 uL in 2X) and purified protein (0.5-10 ug) by SDS-PAGE
-#Analyze uninduced and induced cells (0.5 mL culture resuspended in 100 uL 2X SDS), lysate (5 uL in 2X), unbound (5 uL in 2X) and protein (0.5-10 ug) by SDS-PAGE
 [[Category:Protein Purification]]