To get the animal to react better when creating peptide-antibodies, a carrier protein is crucial. BSA and KLH are two often utilized carrier proteins, with KLH favored due to its increased immunogenicity. Antibodies against both the peptide and the BSA will be produced when peptides and BSA are combined, which could lead to inaccurate ELISA values. We advise include a N terminal cysteine in this reaction because of its great efficiency and demonstrated efficacy in linking carrier proteins to peptides. We use MBS (maleimidobenzoic acid-N-hydroxysuccinimide ester) as a crosslinker for coupling through cysteine. The protein is first activated using MBS, and then the extra crosslinker is removed with SEC. A disulfide bond is then formed between this activated carrier protein and the cysteine in the peptide, with an efficiency of >95%. Salt formation is the only contaminant in this reaction, which we eliminate using gel filtering. We can couple the preferred carrier protein through an amino link in addition to coupling through cysteine. This reaction typically has a coupling efficiency of about 80%. Glutaraldehyde is utilized to crosslink the amino groups in the peptide and carrier protein in order to carry out this process. The possibility of protein-protein and peptide-peptide crosslinking is the reaction's most unpleasant aspect, hence ensuring the right crosslinker concentration is the most crucial step.