Different blocking agents cause variation in the immunologic detection of proteins transferred to nitrocellulose membranes

doi:10.1016/0022-1759(85)90132-2

Journal of Immunological Methods

Volume 81, Issue 1, 16 July 1985, Pages 161-165

https://doi.org/10.1016/0022-1759(85)90132-2 Get rights and content

Abstract

We compared bovine serum albumin, commercial non-fat dry milk, and Tween 20 as blocking agents for immunologic probing of bacterial proteins transferred to nitrocellulose sheets. There were quantitative and qualitative differences in antigens detected that depended on which blocking agents were used. We suggest that several methods for blocking and washing nitrocellulose should be compared when Western blotting is used to detect immunologically reactive proteins.

References (11)

B. Batteiger et al.
J. Immunol. Methods
(1982)
W.N. Burnette
Anal. Biochem.
(1981)
D.A. Johnson et al.
Gene Anal. Techn.
(1984)
H. Towbin et al.
J. Immunol. Methods
(1984)
S.J. Barenkamp et al.
J. Infect. Dis.
(1981)

There are more references available in the full text version of this article.

Cited by (53)

Chapter 33 Performing and Optimizing Western Blots with an Emphasis on Chemiluminescent Detection
2009, Methods in Enzymology
Citation Excerpt :
Many different blocking reagents are available for Western blotting. Because no blocking reagent is appropriate for all systems, empirical testing is essential (Spinola and Cannon, 1985). An optimal blocking buffer maximizes the signal-to-noise ratio and does not react with the system's antibodies or target.
Immunodetection refers to any detection method that exploits the interaction of an antibody and antigen. The choice of detection method, such as enzyme-linked immunosorbent assays (ELISAs) or Western blotting, depends on the researcher's preferences and requirements. If a researcher wants to quantify a low-abundance target protein then a chemiluminescent ELISA is used. If a researcher wants to identify a protein that is in high abundance, a colorimetric Western blot will suffice. If there are multiple targets within an assay, then multiplex fluorescence is typically used.
This article focuses on Western blotting. Although colorimetric and fluorescent detection methods are discussed, chemiluminescent detection is used most often and is, therefore, discussed in great detail. Included is specific information about the chemiluminescent signal and factors that affect its intensity and longevity. We also describe types of blotting and present data and suggestions for obtaining semiquantitative data. Although classical Western blotting is typically used for qualitative purposes, we present information about effective quantitative analysis using specific controls. Common occurrences within the methodology and their possible explanations are also detailed. One frequent result is the appearance of ghost bands, which, based on our research, can be caused by high amounts of target or antibody cross-reactivity. Also included are the basic Western blot protocol and protocols for troubleshooting common problems and optimizing many of the specific factors that influence results.
Western blotting
2006, Methods
Western blotting (protein blotting or immunoblotting) is a powerful and important procedure for the immunodetection of proteins post-electrophoresis, particularly proteins that are of low abundance. Since the inception of the protocol for protein transfer from an electrophoresed gel to a membrane in 1979, protein blotting has evolved greatly. The scientific community is now confronted with a variety of ways and means to carry out this transfer. This review describes the various procedures that have been used to transfer proteins from a gel to a membrane based on the principles of simple diffusion, vacuum-assisted solvent flow and electrophoretic elution. Finally, a brief description of methods generally used to detect antigens on blots is also described.
Blocking nonspecific adsorption of native food-borne microorganisms by immunomagnetic beads with ι-carrageenan
2004, Carbohydrate Research
Citation Excerpt :
Because of the above, we were interested in determining if any blocking agents17 exist that might ameliorate the nonspecific adhesion of bacteria onto IMBs and other capture surfaces. Most blocking agents have been developed for immunoassay applications18–22 and are made from either nonspecific sera, detergents, milk proteins, bovine serum albumin (BSA), ovalbumin, gelatin, or some mixture derived therefrom.17,21,23 These blocking reagents increase an immunoassay’s signal-to-noise ratio by interacting with all solid-phase components and ‘quenching’20 the nonspecific binding of reporter antibodies onto these surfaces.
We present herein the partitioning characteristics of anti-Salmonella and anti-Escherichia coli O157 immunomagnetic beads (IMB) with respect to the nonspecific adsorption of several nontarget food-borne organisms with and without an assortment of well-known blocking agents, such as casein, which have been shown to be useful in other immunochemical applications. We found several common food-borne organisms that strongly interacted with both types of IMB, especially with anti-Salmonella form (av ΔG⁰=−20±4 kJ mol⁻¹) even in the presence of casein [1% (w/v): ΔG⁰=−18±3 kJ mol⁻¹; ΔΔG⁰∼−2 kJ mol⁻¹]. However, when one of the most problematic organisms (a native K12-like E. coli isolate; ΔG⁰=−19±2 kJ mol⁻¹) was tested for nonspecific binding in the presence of ι-carrageenan (0.03–0.05%), there was an average decline of ca. 90% in the equilibrium capture efficiency ξ (ΔG⁰=−11±4 kJ mol⁻¹; ΔΔG⁰∼−8 kJ mol⁻¹). Other anionic polysaccharides (0.1% κ-carrageenan and polygalacturonic acid) had no significant effect (av ΔG⁰=−19±1 kJ mol⁻¹; ΔΔG⁰∼0 kJ mol⁻¹). Varying ι-carrageenan from 0% to 0.02% resulted in ξ significantly diminishing from 0.69 (e.g., 69% of the cells captured; ΔG⁰=−19±3 kJ mol⁻¹) to 0.05 (ΔG⁰=−11±2 kJ mol⁻¹; ΔΔG⁰∼−9 kJ mol⁻¹) at about 0.03% ι-carrageenan where ξ leveled off. An optimum blocking ability was achieved with 0.04% ι-carrageenan suspended in 100 mM phosphate buffer. We also demonstrated that the utilization of ι-carrageenan as a blocking agent causes no great loss in the IMBs capture efficiency with respect to the capture of its target organisms, various salmonellae.
A solid-phase method for evaluation of gold conjugate used in quantitative detection of antigen by immunogold-labeling electron microscopy
2003, Journal of Immunological Methods
Rapid and sensitive screening for confirming the reactivity of reagents, before proceeding for electron microscopy, is highly desirable. ELISA-based methods have been shown to be highly efficient and successful for rapid prescreening and optimization of immunological as well as sample-processing reagents for the sensitive detection and quantitation of antigen by electron microscopy. The drawback of these methods lies in their inability to provide any information regarding the gold conjugate used for the final observed and measured signal. In this work, we demonstrate a simple and rapid, solid-phase method in ELISA format that is also suitable for evaluation and optimization of the gold conjugate. We have demonstrated the utility of this technique by screening for Vitreoscilla hemoglobin (VHb) antigen in cell lysates and confirming the results directly with immunogold-labeling transmission electron microscopy (TEM) of cell sections. The sensitivity of detection and quantitation of antigens by immuno-electron microscopy depends upon the assay procedure being optimized to obtain the best possible signal. Our study indicates that evaluation of gold conjugate by the solid-phase assay could help in the rapid optimization of this reagent for immunogold localization and quantification of antigens by TEM.
Protein blotting: A review
2003, Journal of Immunological Methods
The use of Tween 20 in immunoblotting assays for the detection of autoantibodies in connective tissue diseases
2000, Journal of Immunological Methods
Autoantibodies directed against intracellular antigens can be detected by immunoblotting (IB). Due to its high sensitivity this technique has many advantages, but it can give misleading results when the specific bands are weak or blurred against the background staining. To decrease background staining, non-ionic detergents (Tween 20, Triton X-100, Nonidet P-40) are generally used as blocking agents. Moreover, these agents appear to have a renaturating action towards proteins and antigens. Tween 20 has a more pronounced renaturating effect on proteins than other detergents and thereby improves antigen–antibody binding. To evaluate the effect of Tween 20 on specific autoantibody detection by IB, we tested the sera of 162 patients with connective tissue diseases (CTDs) by adding this detergent at certain steps of the IB assay. We found that the use of Tween 20 in the IB procedure significantly improved the binding of autoantibodies to Jo-1, Scl70, (U1)RNP 68 kDa and C, Sm B/B′ and D. Moreover, it increased the sensitivity for the detection of anti-Sm D peptide in systemic lupus erythematosus (SLE) sera with no decrease in specificity. In contrast, the addition of Tween 20 significantly decreased the binding of autoantibodies specific for ribosomal P proteins, La/SSB, Ro/SSA, but not the overall sensitivity and specificity of the method. We conclude that the addition of Tween 20 to standard IB is advantageous for anti-nuclear antigen antibody detection and improves the sensitivity of the method in revealing anti-Sm-positive sera in SLE. However, Tween 20 is not recommended for the detection of anti-cytoplasmic antibodies.

View all citing articles on Scopus

View full text

Short communicationDifferent blocking agents cause variation in the immunologic detection of proteins transferred to nitrocellulose membranes

Abstract

J. Immunol. Methods

Anal. Biochem.

Gene Anal. Techn.

J. Immunol. Methods

J. Infect. Dis.

Short communication
Different blocking agents cause variation in the immunologic detection of proteins transferred to nitrocellulose membranes