KPL Technical Tips

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Tired of getting poor signal strength and high background in your protein detection applications? Turn your assay around by following the tech tips below to see strong, clear signal and low background in immunoassays such as:

ELISA
Antibodies and Conjugates

Protein Purification

Western Blotting
Immunohistology
Nucleic Acid Labeling and Detection

ELISA

The number and length of washing steps in ELISA can dramatically affect the signal to noise ratio and variability between plates. Visit Critical Factors in Immunoassay Optimization for more details.
Using an automatic plate washer in ELISA can drastically reduce the assay-to-assay variability in background.
Did you know that using chemiluminescence in ELISA is an easy way to increase sensitivity while maintaining low background? Please visit KPL's ELISA Brochure for more information on Chemi-ELISAs.
Direct, Indirect, or Capture (Sandwich) ELISA; which one is best for me? Visit Critical Factors in Immunoassay Optimization to read more about the various types of ELISA and appropriate uses of each.

Western Blotting

When using chemiluminescence for western blotting, conjugate concentration and exposure times are critical factors for achieving optimal signal to noise. To learn more about optimizing background in western blotting, please visit KPL's Western Blot Brochure or Application Note on the use of LumiGLO Reserve in Western Blotting.
Blocking is a critical step in Western Blotting! Careful selection of a blocking system will decrease the likelihood of non-specific background and inhibited signal strength. Learn more by visiting KPL's Blocking Solution Application Note.
Using chemiluminescent substrates in Western blotting can increase sensitivity by a minimum of 10-fold compared to chromogenic substrates! Read more in the LumiGLO Reserve Application Note.
Did you know that milk block solutions are very likley to contain endogenous biotin molecules? This may cause inconsistency and decreased signal strenght in your Biotin-Streptavidin system for Western blotting! Read more about this phenomenon at Blocking Solution Application Note.
Looking for His-tagged proteins? Choosing nickel based detection systems will typically provide greater reliability than using Anti-his antibodies. Check out KPL's Nickel-Labeled Products.

Antibodies and Conjugates

Confused about the type of antibody to use in your immunoassay? Visit Use of Antibodies in Immunoassays for more details.
H+L, Heavy chain Specific, F(ab)₂, Fc, which one is best for your immunoassay? Visit Use of Antibodies in Immunoassays to read about the differences in these antibody formats and the appropriate uses of each format.
Optimizing the antibody dilution in your immunoassay will greatly affect your signal-to-noise ratio.
HRP labeled antibodies have a faster catalytic rate than AP-labeled antibodies. For more information on how this can affect your immunoassay, visit Use of Antibodies in Immunoassays.

Immunohistology

Did you know that tissues may often contain endogenous enzymes which result in non-specific background in IHC? Read more about blocking endogenouse enzymes in tissues at either the Blocking Solution Concentrate or Universal Block Solution product pages.
Blocking steps in IHC typically involve using normal serum derived from the same species used to produce the secondary antibody? Read more about blocking solutions for IHC.
Multiple targets can be easily visualized in one tissue sample. To see how, visit KPL's Double Staining Manual.
Use of TrueBlue Substrate in IHC may increase sensitivity by 10-100 fold from using DAB while also preserving precious primary antibody. Click here to discover TrueBlue!

Protein Purification

Protein G generally exhibits higher affinity to a wider variety of animal species than Protein A. To determine if protein A or G is better for your needs, visit Purification of IgG Molecules.

Nucleic Acid Labeling and Detection

Detection of nucleic acid in non-radioactive systems provides equivalent sensitivity to ³²P use in a shorter amount of time. For a full description of non-rad labeling and detection techniques, visit our Technical Guide.

Did you know that milk block solutions are very likely to contain endogenous biotin molecules? This may cause inconsistency and decreased signal strength in your biotin-Streptavidin system for Northern and Southern blotting! Read more about this phenomenon in our Blocking Solution Application Note.

Using formamide hybridization buffers can drastically improve the specificity of target to probe binding in Northern or Southern blotting. To compare formamide to aqueous buffers, visit our Application Note.

Using RNA probes will provide greater specificity vs.a DNA probe due to the nature of the duplexes formed. Read more about choosing RNA or DNA probes in our Technical Guide.

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