Immunodetection

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Perhaps more than any other step in Western blotting, immunodetection conditions are subject to the greatest number of variables, in part because this step depends on antibody:antigen recognition. Our scientists know how tedious it can be to optimize immunodetection parameters; that’s why we developed the vacuum-driven SNAP i.d.^® Protein Detection System, cutting the time for blocking, probing, and washing to 30 minutes.

Go ahead, troubleshoot – and to learn more about fast immunodetection, click here.

Click on the immunodetection topics to read about the possible causes and remedies:

• Weak Signal
  
• No Signal
  
• Uneven Blot
  
• Speckled Background
  
• High Background
  
• Persistent Background
  
• High Background (Pertains to rapid immunodetection protocol only)
  
• Nonspecific Binding
  
• Reverse Images on Film (White Bands on Dark Background)
  
• Poor Detection of Small Proteins
  
• Presence of Additional Band(s) or Band(s) at the Wrong Size
  
• Large Error Bars in Quantitative Western Blotting

Weak Signal
Possible Cause	Remedy
Improper blocking reagent	The blocking agent may have an affinity for the protein of interest and thus obscure the protein from detection. Try a different blocking agent and/or reduce both the amount or exposure time of the blocking agent. Explore blocking reagents
Insufficient antibody reaction time	Increase the incubation time.
Insufficient signal amplification	Switch from a monoclonal to a polyclonal primary antibody. In polyclonal antibodies, the presence of multiple epitopes on the same protein can generate greater signal. If using a conjugated primary antibody, switch to an unconjugated primary antibody and secondary antibody, which will increase the sensitivity of detection. Biotin-conjugated antibodies provide greater sensitivity and higher amplified signal when compared to fluorochrome- or enzyme-conjugated secondary antibodies. Search for antibodies using our Antibody Finder
Antibody concentration is too low or antibody is inactive	Multiple freeze-thaw cycles, bacterial contamination, or repeated use of antibody solution can change antibody titer or activity. Increase antibody concentration or prepare it fresh. For fluorescent secondary antibodies, ensure that the antibody stock vial and any aliquots are protected from light.
Antibody not suitable for Western blotting or not compatible with preparation of cells/tissue	Check data sheet to see if antibody is compatible with Western blotting. If not, select an alternative antibody. Some antibodies work best on frozen tissue sections, while others prefer fixed tissue. Search for antibodies validated for Western blotting
Outdated detection reagents	Use fresh substrate and store properly. Outdated substrate can reduce sensitivity. Order chemiluminescent substrates
Protein transfer problems	Optimize protein transfer (see above).
Dried blot in chromogenic detection	If there is poor contrast using a chromogenic detection system, the blot may have dried. Try rewetting the blot in water to maximize the contrast.
Tap water inactivates chromogenic detection reagents	Use Milli-Q® water for reagent preparation. Visit our “Water for Western Blotting” learning center
Azide inhibits  HRP	Do not use azide in the blotting solutions.
Antigen concentration is too low	Load more antigen on the gel prior to the blotting.

No Signal
Possible Cause	Remedy
Incompatible primary and/or secondary antibodies	Whenever possible, ensure that the primary antibody is raised against the sequence of the protein found in the same species as your sample. For example, use an antibody targeting the human form of your protein if measuring protein levels in human tissue. If an identical sequence is not possible, determine sequence overlap and reactivity at http://www.ncbi.nlm.nih.gov/protein. Secondary antibodies target the animal species that the primary antibody was raised in. Only use a secondary antibody that was raised in a different species than the host species of the primary antibody, and that is as phylogenetically far from the species of your sample as possible. In addition, ensure that the primary and secondary antibody classes are matched. Most primary antibodies belong to the IgG class, and should be used with a secondary antibody specific for IgG. Search for primary and secondary antibodies
Antibody concentration too low	Increase concentration of primary and secondary antibodies. Search for primary and secondary antibodies
HRP inhibition	HRP-labeled antibodies should not be used in solutions containing sodium azide.
Primary antibody was raised against native protein	Separate proteins in non-denaturing gel or use antibody raised against denatured antigen.
Detection reagent is not sensitive enough	Use a more sensitive detection reagent, with a more appropriate range of detection. Learn about Luminata™ detection reagents for chemiluminescent Western blots Adjust the sensitivity of a Western blot using Luminata chemiluminescent reagents for Western blotting, which provide a wide dynamic range of detection. Order Luminata™ detection reagents

Uneven Blot
	Possible Cause	Remedy
Fingerprints on a Western blot caused by touching membrane without gloves or forceps.	Fingerprints, fold marks or forceps imprints on the blot	Avoid touching or folding membrane; use gloves and blunt end forceps Order blunt-tipped forceps

Speckled Background
	Possible Cause	Remedy
Speckled background on Western blot caused by insufficient washes and/or unfiltered blocking solution.	Aggregates in the blocking reagent	Filter blocking reagent solution through 0.2 µm or 0.45 µm Millex® syringe filter unit. Browse Millex® filters
	Aggregates in HRP-conjugated secondary antibody	Filter secondary antibody solution through 0.2 µm or 0.45 µm Millex® syringe filter unit.

High Background
Overall high background in a Western blot	Possible Cause	Remedy
	Low primary antibody specificity	Switch from a monoclonal to polyclonal primary antibody. The higher specificity of the latter minimizes background. Search for monoclonal and polyclonal antibodies
	Secondary antibody concentration is too high	Increase antibody dilution.
	Secondary antibody cross-reactivity	Use a secondary antibody that has been pre-adsorbed with serum from the species of your target cells or tissue.
	Insufficient washes	Increase washing volumes and times. Explore vacuum-driven SNAP i.d.® system for pulling wash solutions right through the membrane.
	Protein-protein interactions	Use Tween®-20 (0.05%) in the wash and detection solutions to minimize protein-protein interactions and increase the signal to noise ratio.
	Immunodetection on Immobilon®-PSQ transfer membrane	Increase the concentration or volume of the blocking agent used to compensate for the greater surface area of the membrane. Persistent background can be reduced by adding up to 0.5M NaCl and up to 0.2% SDS to the wash buffer and extending the wash time to 2 hours.
	Poor quality reagents	Use high quality reagents and Milli-Q® water. Visit our “Water for Western Blotting” learning center Pre-filter all of your solutions including transfer buffer.
	Cross-reactivity between blocking reagent and antibody	Use different blocking agent or use Tween®-20 detergent in the washing buffer.
	Film overexposure	Shorten exposure time.
	Membrane drying during incubation process	Use volumes sufficient to cover the membrane during incubation.
	Poor quality antibodies	Use high quality affinity purified antibodies.  Search validated, 100% guaranteed antibodies
	Excess detection reagents	Drain blots completely before exposure.
	Detection reagent is too sensitive	Use a less sensitive detection reagent, with a more appropriate range of detection.  Learn about Luminata™ detection reagents for chemiluminescent Western blots

Persistent Background
Possible Cause	Remedy
Nonspecific binding	Use High Salt Wash protocol Adding 0.5 M NaCl and 0.2% SDS to the wash buffer reduced the background considerably on the Immobilon®-PSQ membrane, which has a high protein binding capacity.

High Background (Pertains to rapid immunodetection protocol only )
Possible Cause	Remedy
Membrane wets out during rapid immunodetection	Reduce the Tween®-20 (<0.04%) detergent in the antibody diluent. Use gentler agitation during incubations. Rinse the blot in Milli-Q® water after electrotransfer to remove any residual SDS carried over from the gel. Be sure to dry the blot completely prior to starting any detection protocol.
Membrane was wet in methanol prior to the immunodetection	Do not pre-wet the membrane.
Membrane wasn’t completely dry prior to the immunodetection	Make sure the membrane is completely dry prior to starting the procedure.

Nonspecific Binding
Possible Cause	Remedy
Primary antibody concentration too high	Increase primary antibody dilution. Diluting primary antibody increases specificity.
Secondary antibody concentration too high	Increase secondary antibody dilution.
Antigen concentration too high	Decrease amount of protein loaded on the gel.

Reverse Images on Film (White Bands on Dark Background)
Negative (reverse) image (“bleached” or “burnt-out” bands) on film caused by excess secondary HRP-conjugated antibody (left). The detection was improved by increasing secondary antibody dilution 10-fold (right).	Possible Cause	Remedy
	Too much HRP-conjugated secondary antibody	Reduce concentration of secondary HRP-conjugated antibody.

Poor Detection of Small Proteins
Possible Cause	Remedy
Small proteins are masked by large blocking molecules such as BSA	Consider casein, a low molecular weight blocking agent, such as polyvinylpyrrolidone (PVP), or a protein-free blocking agent Explore blocking reagents Surfactants such as Tween® and Triton® X-100 may have to be minimized. Avoid excessive incubation times with antibody and wash solution.

Presence of Additional Band(s) or Band(s) at the Wrong Size
Possible Cause	Remedy
Nonspecific primary antibody	Verification of antibody specificity is a crucial part of any Western blot experiment. In addition to consulting the antibody data sheet for evidence of specificity, you should conduct your own verification. Common ways to assess specificity include an absence of band(s) in cells/tissue lacking the protein of interest, expected expression in immunohistochemistry, and presence of similar bands using another primary antibody targeting the same protein.
Protein degradation	Ensure adequate protease/phosphatase inhibitor concentration in sample and prepare cell lysate on ice.
Protein glycosylation, phosphorylation/dephosphorylation, ubiquitination, etc. Protein cleavage	A variety of post-translational modifications can change the molecular weight of a protein. Examine the published literature to support this conclusion.

Large Error Bars in Quantitative Western Blotting
Possible Cause	Remedy
Variability in blotting conditions between experimental runs	Ensure identical Western blotting conditions across experiments. Use the same antibody lots for all runs of an experiment. Add more replicates.