Troubleshooting Detection Methods

Extracted from Affinity Chromatography Vol. 2: Tagged Proteins, GE Healthcare, 2016

The troubleshooting guide below addresses problems common to the majority of detection methods as well as problems specific to a particular method. In the latter case, the relevant method is indicated.

Problem Possible Cause Solution
Poor results with the GST Detection Module The 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.
No signal in Western blotting Proteins 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 freshly each time. Store reagents at correct temperature.
Weak signal in  Western blotting Protein 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 blotting Too much protein has been loaded. Reduce the amount of protein loaded.
High backgrounds in Western blotting Washing 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. Use freshly prepared blocking buffer each time.

Increase the concentration of blocking reagent—try 10%.

Use alternative blocking agent (e.g., 1% to 10% BSA, 0.5% to 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 blotting Conjugate 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.


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