| Product Description | PE antibody labeling can be compatible with NaN3, low concentrations of glycerol, Tris, and glycine. The micro ultrafiltration centrifuge tube provided in the kit can quickly remove incompatible small molecule antibody stabilizers before labeling. We also provide enzyme labeled and fluorescently labeled Aladdin ® Kit (please visit www.uelandy.com). Component： 
Matters needing attention： 1. when the tube is taken out of the refrigerator, it should be restored to room temperature first, and then the tube sealing film should be opened to open the product, so as to avoid moisture regain and affect the use effect of the product. Scope of application： Antibody labeling Usage:
The PE antibody labeling kit is suitable for labeling antibodies. We do not recommend labeling other proteins as the labeled DOL (degree of labeling) may not achieve good values.
Check your antibody and antibody compatibility guidelines. If your primary antibody is a commercial product, please contact the supplier for antibody concentration and formula. Antibody solutions without stabilizers can achieve good labeling results, while standard labeling steps are compatible with low concentrations of Tris and glycerol, and the labeling solution is not affected by NaN3. For the standard labeling step (B), label the antibody according to the desired μ Select the specifications of the reagent kit for the quantity of g. To remove non protein components such as Tris, glycine, or glycerol, your antibody can be purified using the ultrafiltration tube provided in the kit, as follows (A).
The antibody concentration with higher labeling efficiency is 1 mg/mL. Ultrafiltration tubes can be used to concentrate antibodies (or clean antibodies). To quantify unknown concentrations of antibodies, UElandy provides WonderOrange ™ Protein quantification kit (W6006) is a highly sensitive protein fluorescence quantification kit. If it is not necessary to remove or concentrate antibodies, follow standard steps (B) for the experiment. 
A. Ultrafiltration steps
Important: Before you start, refer to Table 1 (Antibody Compatibility and Labeling Steps Selection Guide) to determine if your antibodies require ultrafiltration before labeling. If necessary, contact the antibody manufacturer to inquire about the concentration of antibodies and antibody stabilizers. If your antibody does not require ultrafiltration, choose the appropriate labeling step according to Table 1. The molecular weight intercepted by the ultrafiltration membrane is 10 kDa, so molecules smaller than 10 kDa can pass through the membrane, while molecules larger than 10 kDa, including antibodies, will be retained in the ultrafiltration tube (Figure 1). Be careful not to touch the membrane with a straw, as this can tear or puncture the membrane, leading to antibody loss.
Performance of ultrafiltration tubes
Maximum sample volume: 500 μ Final concentrated volume: 15-20 μ L
Volume of filtrate receiver: 500 μ L
Retention volume (membrane/support):<5 μ L
1. Add an appropriate amount of antibodies to the ultrafiltration tube, being careful not to touch the membrane. fourteen thousand × Centrifuge for 2 minutes to filter the liquid into the collection tube and remove the liquid from the collection tube.
For concentrated antibodies, proceed to step 3. For purified antibodies, add an equal volume of 1 × Transfer PBS (pH 7.2) into the ultrafiltration tube. fourteen thousand × Centrifuge for 2 minutes to filter the liquid into the collection tube and remove the liquid from the collection tube. Repeat cleaning 2-3 times.
3. Add an appropriate volume of 1 × Transfer PBS (pH 7.2) into the ultrafiltration tube to achieve a final antibody concentration of 1 mg/mL, and carefully aspirate PBS to resuspend the antibody.
4. Invert the above-mentioned ultrafiltration tube into a new collection tube, 1000 × Centrifuge for 2 minutes and transfer the antibodies from the collection tube to a clean EP tube. 
B. Standard marking steps
Important: Before you start, refer to Table 1 (Antibody Compatibility and Labeling Steps Selection Guide) to determine if your antibody composition and concentration are suitable for labeling with the kit, and which labeling steps are suitable for your selection. If necessary, contact the antibody manufacturer to inquire about the concentration of your antibodies and antibody stabilizers.
1. An antibody concentration of 1 mg/mL is a suitable labeling concentration. If the antibody is in freeze-dried form or at a higher concentration, use 1 × Dissolve or dilute antibodies with PBS (pH 7.2). Transfer the antibody that needs to be labeled into a clean tube. Ensure that the amount of antibody to be labeled matches the labeling range of the reagent kit.
2. Restore the Linking Reagent to room temperature. A brief centrifugation reagent tube concentrates the powder to the bottom.
3. Transfer the antibody (1 mg/mL) to a tube containing Linking Agent, dissolve and mix well, and shake at room temperature (20-25 ℃) for 1 hour.
4. Transfer all the solution from the previous step to the ultrafiltration tube and add 200 μ L 1 × PBS (pH 7.2), 14000 × Centrifuge for 2 minutes. Antibodies will remain in the ultrafiltration tube and the liquid in the collection tube will be discarded.
5. Repeat the cleaning process once.
6. Invert the above-mentioned ultrafiltration tube into a new collection tube, 1000 × Centrifuge for 2 minutes and add an appropriate amount of 1 to the collection tube × PBS (pH 7.2), resuspend the antibody to a final concentration of 1 mg/mL based on the amount of antibody added to the reaction.
7. Transfer the antibodies from the collection tube to a tube containing Modified PE, dissolve and mix well. Incubate at room temperature (20-25 ℃) for 4 hours, avoiding light and shaking.
8. Add 1/10 volume of Storage Buffer to the reaction solution, vortex mix well, and incubate at room temperature (20-25 ℃) in the dark for 1 hour.
9. The modified antibody can be directly used for staining. If it needs to be stored at -20 ℃, an equal volume of glycerol needs to be added. |
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C2416001