Synthesis of NP-Streptavidin Conjugates

4-Hydroxy-3-nitrophenylacetic acid Succinimide Ester (NP-Osu, Biosearch Technologies) was conjugated to Alexa Fluor 647 Streptavidin (SA-AF647) or Alexa Fluor 594 Streptavidin at a hapten:streptavidin molar ratio of 10:1 or 20:1. Biotinylated Eα_52–73 peptide (N-biotin-GSGFAKFASFEAQGALANIAVDKA-COOH)^{15 (link),77 (link)} was synthesized at the RU Proteomics Resource Center. NP-Streptavidin conjugates were incubated with a 6x molar excess of biotinylated Eα peptide, and excess peptide was removed by dialysis. Hapten-protein conjugation ratios were calculated by measuring the absorbance value at 430 nm. For αDEC-OVA-Eα experiments shown in Figures S4E and S4F, NP-SA-AF647 conjugates were incubated with a 30x molar excess of D-biotin and excess D-biotin was removed by dialysis.

Free full text: Click here

Chen S.T., Oliveira T.Y., Gazumyan A., Cipolla M, & Nussenzweig M.C. (2023). B cell receptor signaling in germinal centers prolongs survival and primes B cells for selection. Immunity, 56(3), 547-561.e7.

Publication 2023

Succinimide Ester NP-Osu

4 hydroxy 3 nitrophenylacetic acid Af647 Alexa 594 Biotin Dialysis Ester Hapten Molar Peptide Protein Streptavidin Succinimide

Corresponding Organization : Howard Hughes Medical Institute

Top 5 similar protocols

Variable analysis

independent variables

Hapten:streptavidin molar ratio (10:1 or 20:1)

dependent variables

Hapten-protein conjugation ratios

control variables

Biotinylated Eα peptide (N-biotin-GSGFAKFASFEAQGALANIAVDKA-COOH)
NP-Streptavidin conjugates
Excess D-biotin

controls

Positive control: None specified
Negative control: None specified

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!