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Troubleshooting Detection Methods for GST-tagged Proteins

This troubleshooting guide addresses problems common to many of the detection methods for GST-tagged proteins and problems specific to a particular method.

ProblemPossible CauseSolution
Poor results with the GST Detection ModuleThe reaction rate is nonlinear.The reaction rate of the CDNB assay is linear provided that an A340 of ~ 0.8 is not exceeded during the 5-min time course.
Plot initial results to verify that the reaction rate is linear over the time course. Adjust the amount of sample containing the GST-tagged protein to maintain a linear reaction rate.
The target protein has inhibited the folding of the GST tag.The tagged protein may have inhibited the correct folding of the GST moiety. The GST-tagged proteins will thus show very low activity with the CDNB assay. Whether for this or for any other reason, if a low absorbance is obtained using the CDNB assay, a Western blot using anti-GST antibody may reveal high levels of tagged protein expression.
There is baseline drift.Under standard assay conditions at 22 °C and in the absence of GST, glutathione and CDNB react spontaneously to form a chemical moiety that produces a baseline drift at ∆A340/min of ~ 0.003 (or 0.015 in 5 min). Correct for baseline drift by blanking the spectrophotometer with the blank cuvette before each reading of the sample cuvette. Alternatively, get the slope directly from the spectrophotometer software. The slope will be the same as long as the spontaneous reaction is limited.
Poor results with the GST 96-Well Detection ModuleLow absorbance is seen in the assay.Check that host cells were sufficiently induced, that the samples were sufficiently lysed, and that inclusion bodies have not been formed.
Concentration of blocking buffer is inadequate.If clarified lysate is being tested, mix the initial GST sample with 2X blocking buffer to give a final concentration of 1X blocking buffer.
There is poor day-to-day reproducibility.Verify that all incubation times are consistent. GST capture incubation time can be decreased with slightly reduced signal, but do not incubate for less than 30 min. Every 15-min decrease in HRP/anti-GST conjugate incubation time can significantly reduce signal.
No signal in Western blottingProteins are not transferred during Western blotting.Stain gel and membrane with total protein stain to check transfer efficiency. Optimize gel acrylamide concentration, time for transfer, and current.
Ensure gel and membrane make proper contact during blotting and are orientated correctly with respect to the anode.
Check that excess temperatures are not reached during electroblotting, producing bubbles or membrane distortion.
Proteins are not retained on membrane.Assess transfer of proteins (as above). Use a fresh supply of membrane.
There are problems with detection reagents.Ensure reagents are being used correctly. Prepare reagents just before use each time. Store stock reagents at correct temperature.
Weak signal in Western blottingProtein transfer efficiency is poor.Check transfer efficiency as above.
Insufficient protein has been loaded.Load more protein on gel.
Exposure time is too short.Increase exposure time.
Excessive diffuse signal in Western blottingToo much protein has been loaded.Reduce the amount of protein loaded.
High backgrounds in Western blottingWashing is inadequate.Ensure post-conjugate washes are performed for a sufficient amount of time with an adequate volume of wash buffer (> 4 mL/cm2 membrane).
Blocking is inadequate.Check the blocking buffer has been made correctly. Prepare blocking buffer just before use each time.
Increase the concentration of blocking reagent—try 10%.
Use alternative blocking agent (e.g., 1-10% BSA, 0.5-3% gelatin).
Increase incubation time with blocking buffer.
Blotting equipment or buffers are contaminated.Clean equipment. Prepare fresh buffers.
Multiple bands are seen in Western blottingConjugate is binding non-specifically to other proteins.Include a negative control of expression host not containing expression vector to determine nonspecific binding.
GST-tagged protein may have been degraded.Include protease inhibitors during purification. Reduce purification time and temperature. Add a second purification step to remove incomplete target protein.

Materials

Product NumberProduct NameProduct DescriptionPricing
GERPN800EAmersham ECL Rainbow Marker - Full rangeFull Range, Cytiva RPN800E
GERPN756EAmersham ECL Rainbow Marker - Full rangeHigh Range, Cytiva RPN756E
GERPN755EAmersham ECL Rainbow Marker - Full rangeLow Range, Cytiva RPN755E
GE10600121Amersham Hybond® P Western blotting membrane sandwich, PVDFpore size 0.45 μm, sheet W × L 80 mm × 90 mm , preassembled with 2 x 3MM chr filter papers, pkg of 10 ea
GE10600122Amersham Hybond® P Western blotting membrane sandwich, PVDFpore size 0.2 μm, sheet W × L 80 mm × 90 mm , preassembled with 2 x 3MM chr filter papers, pkg of 10 ea
GE10600023Amersham Hybond® P Western blotting membranes, PVDFpore size 0.45 μm, roll W × L 300 mm × 4 m, pkg of 1 ea
GE10600021Amersham Hybond® P Western blotting membranes, PVDFpore size 0.2 μm, roll W × L 260 mm × 4 m, pkg of 1 ea
GE10600029Amersham Hybond® P Western blotting membranes, PVDFpore size 0.45 μm, roll W × L 150 mm × 4 m, pkg of 1 ea
GE10600038Amersham Hybond® P Western blotting membranes, PVDFpore size 0.45 μm, sheet W × L 300 mm × 600 mm, pkg of 5 ea
GE10600058Amersham Hybond® P Western blotting membranes, PVDFpore size 0.2 μm, sheet W × L 200 mm × 200 mm, pkg of 10 ea
GE10600057Amersham Hybond® P Western blotting membranes, PVDFpore size 0.2 μm, sheet W × L 200 mm × 200 mm, pkg of 25 ea
GE10600069Amersham Hybond® P Western blotting membranes, PVDFpore size 0.45 μm, sheet W × L 140 mm × 160 mm, pkg of 25 ea
GE10600087Amersham Hybond® P Western blotting membranes, PVDFpore size 0.45 μm, sheet W × L 100 mm × 100 mm, pkg of 10 ea
GE10600086Amersham Hybond® P Western blotting membranes, PVDFpore size 0.45 μm, sheet W × L 100 mm × 100 mm, pkg of 25 ea
GE10600101Amersham Hybond® P Western blotting membranes, PVDFpore size 0.2 μm, sheet W × L 80 mm × 90 mm, pkg of 25 ea
GE10600100Amersham Hybond® P Western blotting membranes, PVDFpore size 0.45 μm, sheet W × L 80 mm × 90 mm, pkg of 25 ea
GERPN2109ECL Blotting ReagentsCytiva RPN2109
GERPN810ECL DualVue Western MarkersCytiva RPN810, sufficient for 25 blots
GERPN2236ECL Prime Western Blotting Detection ReagentCytiva RPN2236
GERPN2232ECL Prime Western Blotting SystemCytiva RPN2232
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