5.1 Formulations of Reagents to Be Prepared for Antibody Incubation

Reagent Formulations
1. Antibody Diluent
First, prepare a 5% BSA (W/V, i.e., add 5 g of BSA to 100 mL of solution) using TBST. This serves as the antibody diluent for later use.
2. Preparation of Primary Antibody
Dilute the primary antibody according to the ratio specified in the instruction manual. For example, for a 1:1000 dilution to prepare 10 mL of antibody working solution: add 10 μL of undiluted primary antibody to 10 mL of the 5% BSA antibody diluent.
If the antibody is to be reused multiple times, check whether sodium azide has been added to the undiluted primary antibody. If not, approximately 0.01% sodium azide can be added to the diluted antibody as a preservative.
3. Preparation of Secondary Antibody
Dilute the secondary antibody in the prepared 5% BSA solution according to the ratio in the instruction manual. For example, for a 1:1000 dilution to prepare 10 mL of antibody working solution: add 10 μL of undiluted secondary antibody to 10 mL of the 5% BSA antibody diluent.
★ If the purchased secondary antibody is in powder form, it needs to be reconstituted and diluted in advance. For reconstitution and storage methods, refer to Jackson’s guidelines for reconstitution and storage of secondary antibodies:
(1) HRP-conjugated secondary antibody: Do not contain sodium azide (a preservative) (sodium azide will interfere with HRP activity).
(2) Fluorophore-conjugated secondary antibody: Contains 0.05% sodium azide as a preservative.
Reconstitution and Storage:
Store the lyophilized powder at 2–8°C.
When ready to use, reconstitute the lyophilized antibody with a specific volume of dH₂O (distilled water) (the reconstitution volume varies by antibody; follow the instruction manual). If turbidity persists after thorough dissolution, centrifuge the solution.
After reconstitution, the antibody is stable at 2–8°C for 6 weeks. For each use, freshly dilute it to the required working concentration.
To extend the storage period, aliquot the reconstituted antibody and store it at -70°C or below. Avoid repeated freezing and thawing. Alternatively, add an equal volume of ACS-grade (or higher) glycerol to adjust the antibody concentration to 50% of the reconstituted concentration, then store at -20°C.
Note: Adding glycerol halves both the protein concentration and the dilution range. For example: If the reconstituted antibody has a concentration of 0.8 mg/mL and a volume of 2 mL, with a recommended dilution ratio of 1:5,000–1:100,000 for WB. After adding an equal volume (2 mL) of glycerol, the antibody concentration becomes 0.4 mg/mL, the volume becomes 4 mL, and the dilution ratio for WB adjusts to 1:2,500–1:50,000.
Validity Period: 1 year after reconstitution. If the experimental results remain within an acceptable range beyond the validity period, the antibody can still be used.
Caution: Ensure the glycerol is ACS-grade or higher. Glycerol below ACS grade may severely impair the activity of peroxidase.
4. Preparation of Developing Solution
After incubating the membrane with the secondary antibody and completing the washing steps, prepare the developing solution by mixing Solution A and Solution B at a 1:1 ratio. Allow the mixture to stand at room temperature for subsequent development.