Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 59)

Total Protein Staining
Problem Cause Solution
Spots not visible (see 2-D No protein in the gel Use another staining method to confirm
Gel Evaluation, below) that there is protein in the gel
Malfunctioning imaging Check the instrument manual for
system or incorrect troubleshooting information, or contact
imaging parameters the imaging instrument manufacturer
Poor staining Insufficient protein in the gel Repeat the experiment with a
load sensitivity higher protein quantity
Dirty staining trays (for example, Clean the staining trays and other
with silver staining) equipment thoroughly with laboratory
glassware cleaner
Insufficient stain volume Follow the recommendations for stain
volume appropriate to the gel size
Insufficient staining time Increase staining time
Reuse of staining solution To ensure quantitative reproducibility
of a 2-D experiment, never reuse
staining solution
High or uneven Dirty equipment or staining trays Clean the staining trays and other equipment
background staining thoroughly with laboratory glassware cleaner
Too much time in staining solution Restrict the time in staining solution
as recommended
Wash the gel in water or respective
destaining solution for >30 min
Reagent impurities Make sure that the water and reagents
used for staining are of the highest
possible quality
Diffuse, uneven Insufficient washing Perform more washing steps. Use purified
background in laboratory water and clean staining trays
silver-stained gel
Do not place too many gels in one tray.
Fully immerse the gels in the staining
solution; they should not stick to the
staining tray
Insufficient fixative (some uneven Apply a longer fixing procedure
background stain is normal when
using a silver stain. Due to migration
of different chemicals and ions
into the gels, some regions can
be stained with different colors
or intensities)
Contaminant(s) in the agarose Prepare fresh overlay solution
overlay solution
Total Protein Staining (contd.)
Problem Cause Solution
Speckles or blotches in Particulate material from reagents, Clean the staining trays and other
the gel image staining tray, dust, or gloves equipment thoroughly with laboratory
glassware cleaner
Limit exposure of gels and staining
solution to open air
Use dust-free gloves, and handle gels
only by the edges
Uneven staining Insufficient shaking during staining Agitate the gel during staining
Gel shrinkage Some gel shrinkage occurs Transfer the gel to water
during staining
2-D Gel Evaluation*
Problem Cause Solution
No Spots or Fewer Spots
than Expected
Across the gel Insufficient sample was loaded Check the sample concentration by
protein assay
Check that the protein assay is functioning
properly and that it is not responding to
interfering substances in your sample
Insufficient sample entered Start IEF at a low field strength
the IPG strip
Make sure that the IPG strips are in the
correct orientation in the focusing tray
Check that the orientation of
electrical connections
Increase the solubility strength of the
2-D sample solution; insoluble proteins
will not enter the IPG strip
Failure of detection reagents Run a lane of unstained standards adjacent
to the second-dimension separation. If the
standards are not detected, check the
expiration dates and the formulations of
all detection reagents
Staining method not See Chapter 3 for sample loading
sensitive enough recommendations dependent on the
staining technique used
Poor protein transfer from Perform the first stage of SDS-PAGE at
IPG strip to SDS-gel low voltage (50 V) for >20 min until the
bromophenol blue front enters the
separation gel (time depends on gel size)
* Also refer to Berkelman et al. (2004) and Bio-Rad Laboratories (2005).
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2-D Electrophoresis Guide Troubleshooting