Human Histone H1.2 peptide (ab16936)

Thank you for your e-mail. We are very pleased to hear that you are satisfied with the antibody ab4086. I enclose our recommended protocol for blocking, please do not hesitate to contact us again if you need further information. In principle, a small volume of antibody (e.g., 1-5 ul) is first reacted with excess peptide (5-50 fold over the antibody; e.g., 1 ug antibody reacted with 5-50 ug peptide; exact amounts determined by titration) to neutralize it. The neutralized antibody can no longer subsequently bind to another antigen (a band of interest) or staining pattern. So the band(s)/staining that is competed by the antigen/peptide is supposedly specific. If more than one band disappears by peptide/antigen competition then those bands have the antigenic determinants and could be considered either fragments of the large antigen or multimer. 1. Determine the amount of antibody that is needed for 1 strip (e.g., 2 ml). For example, an antibody has given desired bands at 1:1000 dilution. So you will need 1 ul/ml antibody (2-ul antibody for 2-ml antibody solution). If an antibody were used at 1:5000 dilution then you would only need 0.2 ul/ml (use 2 ul of 1:10 dilution for better accuracy). 2. Take 2-ul antibody (or as needed) in 100 ul saline/PBS. Make 2 tubes. Add antigen/peptide solution (10-50 ug peptide or antigen added in 10-100 ul). Add same volume of saline/PBS (no peptide/antigen) to the other tube labeled as -peptide or No peptide. Mix gently. 3. Incubate both tubes at 37oC for 1-2 hrs or 2-24 hrs at 4oC. 4. Centrifuge the tubes for 15 min at 4oC in a microfuge (10-15000 rpm) to pellet any immune complexes. Carefully remove the supernatant. If no visible pellet is seen than just leave approx. 5-10 ul at the bottom to avoid disturbing invisible immune complexes. If you do not centrifuge the solution, it may give high background. 5. After centrifugation, make up the volume of supt. to 2 ml (or what is necessary depending upon the initial antibody taken) with buffer (PBS-Tween with or without BSA or milk or any buffer that was used initially). Use both antibodies (with and without antigen/peptide) for Western blotting or immunolocalization. 4. Observe the bands/staining that disappears. We have tested ab4086 specificity by mixing it at 1/500 with 1µg/ml of peptide Histone H1.2 ab16936.