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Synthesis of Peptide Arrays Using SPOT-Technology and ..

Antibody binding was visualized using a secondary antibody and ECL detection system. To avoid overestimation of cross-reactivity and potential saturation of the detection system, antibodies with high levels of cross-reactivity or high signal intensity were assayed at higher dilutions until the results no longer changed with further dilution. For quality control, each glass slide contained two copies of the array. Binding to these internal duplicates was highly reproducible, which ensures reproducible spotting (). Binding to slides with independently synthesized peptides was found to be very similar as well (). We compared the binding of antibodies to different peptides containing the primary epitope (by excluding the false negatives, see below) and observed very reproducible binding intensities with standard errors within 5–30% while the difference between bound spots and background was usually more than 100-fold (). These observations indicated that the synthesis of the peptides was comparable between different peptides and between batches and the signal/noise ratio of the readout is excellent.

Synthesis of Peptide Arrays Using SPOT-Technology and the ..

To confirm our data, we have performed a dot blot analysis of selected antibodies using purified peptides (). The H3K9me3 antibody #11 showed weaker binding to H3K9me3/S10ph than to H3K9me3, while antibody #10 bound both peptides equally. Furthermore, H3K9me3 antibody #9 showed cross-reactivity with H4K20me3 and H3K27me3. The H3K27me3 antibody #20 showed cross-reactivity with H3K9me3 and H4K20me3. Finally, the H4K20me3 antibody #33 did not show binding of H3K27me3 or H3K9me3. These results qualitatively agree with the data from the Celluspots arrays indicating that Celluspots arrays are a reliable tool to study the binding specificities of the antibodies.

Synthesis of Peptide Arrays Using SPOT-Technology ..

A Novel Multipeptide Microarray for the Specific and ..

Chromatin structure is greatly influenced by histone tail post-translational modifications (PTM), which also play a central role in epigenetic processes. Antibodies against modified histone tails are central research reagents in chromatin biology and molecular epigenetics. We applied Celluspots peptide arrays for the specificity analysis of 36 commercial antibodies from different suppliers, which are directed towards modified histone tails. The arrays contained 384 peptides from eight different regions of the N-terminal tails of histones, viz. H3 1–19, 7–26, 16–35 and 26–45, H4 1–19 and 11–30, H2A 1–19 and H2B 1–19, featuring 59 post-translational modifications in many different combinations. Using various controls we document the reliability of the method. Our analysis revealed previously undocumented details in the specificity profiles of the tested antibodies. Most of the antibodies bound well to the PTM they have been raised for, but some failed. In addition, some antibodies showed high cross-reactivity and most antibodies were inhibited by specific additional PTMs close to the primary one. Furthermore, specificity profiles for antibodies directed toward the same modification sometimes were very different. The specificity of antibodies used in epigenetic research is an important issue. We provide a catalog of antibody specificity profiles for 36 widely used commercial histone tail PTM antibodies. Better knowledge about the specificity profiles of antibodies will enable researchers to implement necessary control experiments in biological studies and allow more reliable interpretation of biological experiments using these antibodies.

Peptide arrays synthesized on cellulose membranes by the SPOT method were introduced by R. Frank in 1992. Since that time, peptide arrays developed into valuable tools that were applied, for example, to analyze the specificity of peptide modifying enzymes and the binding specificities of antibodies and proteins including epigenetic reading domains.,,,, Here, we used Celluspots peptide arrays comprising 384 peptides to study the specificity of the interaction of 36 different commercial antibodies raised for modified histone tails. In the Celluspots technique, peptides are synthesized on cellulose support as in conventional SPOT synthesis, but after synthesis the cellulose matrix together with the peptides is solubilized and spotted on glass slides. Due to miniaturization when compared with conventional SPOT synthesis, Celluspots arrays can be produced at lower costs and assays can be performed using much less reagent. The arrays contained peptides from eight different regions of the N-terminal tails of histones, viz. H3 1–19, 7–26, 16–35 and 26–45, H4 1–19 and 11–30, H2A 1–19 and H2B 1–19 ( and ), featuring 59 post-translational modifications (most of them identified, some hypothetical) in many different combinations.

using SPOT-technology and the CelluSpots method.

01/10/2015 · Detailed specificity analysis of antibodies binding to ..

Celluspots arrays spotted on glass slides were prepared as described. They are now commercially available from Active Motif (Cat. No. 13001). To confirm the identity of the peptides, analysis representative peptides were synthesized in parallel starting with an acid labile Rink linker and cleaved off from the matrix and subjected to mass spectrometric characterization (). For antibody binding, the array was blocked by incubation in TTBS buffer (10 mM Tris/HCl pH 7.5, 0.05% Tween-20 and 150 mM NaCl) containing 5% non-fat milk powder at 4°C overnight, then washed three times with TTBS buffer and incubated at room temperature for 1 h with antibody dilutions as recommended by the supplier for western blot staining. Antibodies were dissolved in 2 ml of TTBS buffer. In case of high cross-reactivity or strong signal, antibody concentrations were decreased stepwise until results were stable with further dilution. Final antibody concentrations used are given in . The array was washed three times with TTBS and incubated with horseradish peroxidase conjugated anti-rabbit or anti-mouse antibody (GE Healthcare, 1:2,500) in TTBS for 1 h at room temperature. Finally, the array was washed three times with TTBS and submerged in ECL developing solution (Thermo Fisher Scientific) and the image was captured in X-ray film. To avoid saturation of the ECL detection system and the X-ray film, all images were developed several times with different exposition and only images which were not saturated were used for the analysis. Typical exposure times were 0.5–5 min. Analysis was done using an in house program (Array Analyze, available at or from the authors upon request). False positives were identified by manual inspection and by calculation of the average binding intensity to all spots carrying a particular modification. False negatives were identified by manual inspection. For quantitative description of the quality of an antibody, we use the specificity factor, which is defined as the ratio of the average binding intensity to all peptides carrying a particular modification and the average binding intensity to all peptides not having that modification. It gives an overall measure of the binding specificity of the antibody taking into account false positives and negatives. The antibodies used in this study were arbitrarily chosen to represent different positions on the histone tails and types of modifications. Different batches of the same antibody may yield different results.

Chromatin structure is greatly influenced by histone tail post-translational modifications (PTM), which also play a central role in epigenetic processes. Antibodies against modified histone tails are central research reagents in chromatin biology and molecular epigenetics. We applied Celluspots peptide arrays for the specificity analysis of 36 commercial antibodies from different suppliers, which are directed towards modified histone tails. The arrays contained 384 peptides from eight different regions of the N-terminal tails of histones, viz. H3 1–19, 7–26, 16–35 and 26–45, H4 1–19 and 11–30, H2A 1–19 and H2B 1–19, featuring 59 post-translational modifications in many different combinations. Using various controls we document the reliability of the method. Our analysis revealed previously undocumented details in the specificity profiles of the tested antibodies. Most of the antibodies bound well to the PTM they have been raised for, but some failed. In addition, some antibodies showed high cross-reactivity and most antibodies were inhibited by specific additional PTMs close to the primary one. Furthermore, specificity profiles for antibodies directed toward the same modification sometimes were very different. The specificity of antibodies used in epigenetic research is an important issue. We provide a catalog of antibody specificity profiles for 36 widely used commercial histone tail PTM antibodies. Better knowledge about the specificity profiles of antibodies will enable researchers to implement necessary control experiments in biological studies and allow more reliable interpretation of biological experiments using these antibodies.

SPOT synthesis, Celluspots arrays can ..
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Therefore, taking peptide arrays ..

To confirm our data, we have performed a dot blot analysis of selected antibodies using purified peptides (). The H3K9me3 antibody #11 showed weaker binding to H3K9me3/S10ph than to H3K9me3, while antibody #10 bound both peptides equally. Furthermore, H3K9me3 antibody #9 showed cross-reactivity with H4K20me3 and H3K27me3. The H3K27me3 antibody #20 showed cross-reactivity with H3K9me3 and H4K20me3. Finally, the H4K20me3 antibody #33 did not show binding of H3K27me3 or H3K9me3. These results qualitatively agree with the data from the Celluspots arrays indicating that Celluspots arrays are a reliable tool to study the binding specificities of the antibodies.

Peptide Microarrays von Marina Cretich | ISBN 978-1 …

Altogether, we tested the peptide tail binding specificity of 36 antibodies from major suppliers and analyzed the results in detail ( and , examples of the results are shown in , all technical details are given in , the tables of spot intensities are provided in ). In most cases the antibodies showed highest specificity towards the expected target although some of them failed (like #25), which is critical for previous studies using these reagents. Furthermore, our data revealed important details on the specificity of the antibodies which were not documented before. For example, antibody #5 directed against H3K4me3 showed weak binding to peptides also containing H3T3ph (false negatives) and cross-reactivity at some H4K20me3 spots (false positives) (). Antibody #9 directed against H3K9me3 bound weakly to peptides also containing H3S10ph or H3T11ph and it showed cross-reactivity at H3K27me3 and H4K20me3. The different “pan”-antibodies supposed to bind to a defined set of related modifications were particular critical cases, since all of them showed clear preferences; sometimes the binding spectrum was rather restricted. The importance of an antibody specificity analysis can be further exemplified by the results obtained with four different H3K27me3 antibodies (). While all of them bind to the single modified H3K27me3 peptide with good preference, antibody #19 did not bind to any of the peptides carrying additional modifications like R26me2a, R26me2s (which is hypothetical at present) or S28ph and it showed weak cross-reactivity at H3K27ac. Antibody #20 bound to all H3K27me3 peptides but it showed strong cross-reactivity at H4K20me3 and weak cross-reactivity at H3K4m3 and H3K9me3. Antibody #21 did not show cross-reactivity, but it did not bind to peptides containing S28ph in addition to K27me3. Antibody #22 showed cross-reactivity at H4K20me3 and showed only weak binding to peptides containing S28ph in addition to K27me3. Therefore, studies on H3K27me3 distribution likely will come to different conclusions if these different antibodies are used.

Web-Based Design of Peptide Microarrays Using ..

Altogether, we tested the peptide tail binding specificity of 36 antibodies from major suppliers and analyzed the results in detail ( and , examples of the results are shown in , all technical details are given in , the tables of spot intensities are provided in ). In most cases the antibodies showed highest specificity towards the expected target although some of them failed (like #25), which is critical for previous studies using these reagents. Furthermore, our data revealed important details on the specificity of the antibodies which were not documented before. For example, antibody #5 directed against H3K4me3 showed weak binding to peptides also containing H3T3ph (false negatives) and cross-reactivity at some H4K20me3 spots (false positives) (). Antibody #9 directed against H3K9me3 bound weakly to peptides also containing H3S10ph or H3T11ph and it showed cross-reactivity at H3K27me3 and H4K20me3. The different “pan”-antibodies supposed to bind to a defined set of related modifications were particular critical cases, since all of them showed clear preferences; sometimes the binding spectrum was rather restricted. The importance of an antibody specificity analysis can be further exemplified by the results obtained with four different H3K27me3 antibodies (). While all of them bind to the single modified H3K27me3 peptide with good preference, antibody #19 did not bind to any of the peptides carrying additional modifications like R26me2a, R26me2s (which is hypothetical at present) or S28ph and it showed weak cross-reactivity at H3K27ac. Antibody #20 bound to all H3K27me3 peptides but it showed strong cross-reactivity at H4K20me3 and weak cross-reactivity at H3K4m3 and H3K9me3. Antibody #21 did not show cross-reactivity, but it did not bind to peptides containing S28ph in addition to K27me3. Antibody #22 showed cross-reactivity at H4K20me3 and showed only weak binding to peptides containing S28ph in addition to K27me3. Therefore, studies on H3K27me3 distribution likely will come to different conclusions if these different antibodies are used.

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