Cell or tissue type does not express the protein of interest
Perform a positive controlPreferably from cell or tissue lysate already verified to express the target protein
Improper cell treatment
Stimulate cells with the appropriate chemical, protein, etc.Verify that the stimulation works
Improper sample preparation for gel loading
Ensure that the protein in the lysate is stable.Use appropriate protease and phosphatase inhibitors, etcEnsure protein samples contain SDS, and have been heated prior to gel loadingInclude a reducing agent such as dithiothreitol (DTT) and/or 2-mercaptoethanol
Specific antigen concentration is too low
Load more protein on gelEnrich the antigen by fractionation or by immunoprecipitation
Proteins did not transfer properly to the membrane
Wet PVDF membrane in methanol or nitrocellulose membrane in transfer buffer before useEnsure there is good contact between the membrane and the gelOptimize the transfer timeAfter transfer, ensure molecular weight markers were transferredStain the membrane with Ponceau red, and the gel with Coomassie blue
Insufficient antigen binding to membrane
If the antigen has low molecular mass, it may pass through the membraneSwitch to a membrane with a smaller pore sizeSwitch to a different type of membrane
The antigen is masked by the blocking buffer
Test different blocking buffersTry milk, serum, BSA in Tris-buffered saline & PBSTest different concentrations of each
Insufficient amount of antibodies present
Increase concentration of primary and/or secondary antibody
Antibody exposure time is too short
Increase the exposure time
Antibodies may have lost activity
Test antibodies by performing a Dot Blot
Excessive washing of the membrane
Reduce the number of washes
Substrate incubation is too short
Increase substrate incubation time
Substrate has lost activity
Test substrate using a positive control
Na azide inhibits enzyme reaction of HRP-conjugated Ab
Do not use sodium azide together with HRP-conjugated antibodies
Possible Cause
Solution
Non-specific antibody binding
Reduce primary antibody concentrationDecrease the amount of total protein loaded on gelAdjust membrane blocking conditionsIncrease number of washesVerify the specificity of the antibodyBlot with the secondary antibody alone.If bands develop, choose an alternate secondary antibody
Degradation of protein
Prepare fresh samplesUse protease inhibitors during sample preparationMinimize freeze/thaw cycles of sample
Aggregation of analyte
Increase the amount of DTT (20 -100mM) to ensure complete reduction of disulfide bondsHeat in boiling water bath for 5-10 minutes before loading onto gel
Cell lines have been passaged extensivelyDifferences in protein expression profiles result
Go back to the original non-passaged cell lineRun the current and original cell line samples side-by-side
Protein has multiple modifications in vivoAcetylation, methylation, glycosylation, phosphorylation, etc
Review the literature for modified protein variantsAdjust sample preparation accordingly
Target protein has multiple isoformsOther proteins share similar epitopes
Check the literature for target protein isoformsPerform a BLAST search to check for possible cross-reactionsInclude other cell or tissue types
Possible Cause
Solution
Too much protein per lane
Titrate down the amount of protein loaded per lane
Insufficient blocking of non-specific binding
Adjust blocking conditionsInclude blocking agent in the antibody buffers as well
The primary antibody concentration may be too high
Titrate the antibody to find the optimal concentration
The secondary antibody may be binding non-specifically
Blot with the secondary antibody aloneIf bands develop, choose an alternate secondary antibody
Incubation temperature may be too high
Incubate blot at 4°C
Cross-Rxn between blocking agent & primary or secondary AbAntibody cross-reacts with casein, a milk phosphoprotein
Add a mild detergent, e.g.Tween® 20, to incubation & washing buffersRecommended for phosphoprotein specific antibodiesUse BSA as a blocking reagent instead of milk
Washing of unbound antibodies may be insufficient
Increase the number of washes
The membrane may give high background
Nitrocellulose membrane may give less background than PVDF
The membrane has dried out
Avoid drying out the membrane during processing and incubation