Common Questions and Answers for Labeling Kits
Q1:What types of antibodies or proteins can be labeled with small molecule dye labeling kits?
A:Conventional IgG from different species can be labeled. IgM antibodies can also be labeled with appropriate adjustments in dye dosage or labeling buffer. Other antibody types such as IgY, IgD, and IgA have not been validated, but theoretically can be labeled as long as there are free primary amine groups on the antibody or protein.
Q2:What are the considerations for the material to be labeled?
A:The material to be labeled must have free primary amines or N-termini. The sample should not contain Tris, glycerol, proteins or reagents with amino groups.
Q3:Can small or trace amounts of labeling be performed (e.g., less than 10 μg of antibody protein)?
A:The current method in the manual is for labeling 0.02-2 mg of antibody. For smaller amounts, we can provide customization or labeling services. We will also launch a kit for labeling less than 10 μg of antibody protein in the future.
Q4:Can a certain substance (e.g., folic acid/peptide) be labeled with FITC/Fluor594 using the labeling kit?
A:If a substance has free primary amine groups, it can theoretically be labeled. For proteins, the number of primary amines (lysine) should be considered. For small molecules, the number of amines determines the labeling. Folic acid has one amino group on its nitrogen-containing heterocycle, which can theoretically be labeled with FITC or Fluor594. However, the nucleophilicity of this amino group is relatively weak, and the labeling efficiency may not be good. Additionally, the size of the labeled substance should match the appropriate ultrafiltration tube for purification, which the customer needs to decide based on their experimental purpose.
Q5:How to improve the labeling efficiency (DOL) of biotin/small molecule fluorescent dyes?
A:There are many ways to give it a try.
1) Increase the ratio of dye to protein in the reaction.
2) Increase the protein concentration in the reaction system.
3) Appropriately increase the reaction temperature and time.
4) Change the labeling buffer for the target protein.
Q6:Does higher labeling efficiency always lead to better experimental results?
A:No. Generally, for conventional IgG antibodies, a DOL between 2 and 9 is more appropriate. If the DOL exceeds this range, the experimental results usually deteriorate. The specific experimental results depend on the characteristics of the protein and antibody being labeled, and the optimal DOL should be determined based on actual experimental results.
Q7:How to quantify and determine the concentration of recombinant proteins after labeling with small molecule dyes?
A:There are various methods for quantifying recombinant proteins, such as BCA method, UV absorption method, Bradford, Coomassie Brilliant Blue, Lowry method, etc., to measure protein concentration. Alternatively, the absorbance can be measured according to the manual to calculate the concentration. Each method has its limitations, and customers can choose the appropriate measurement scheme based on their actual situation.
Q8:Can biotin labeling kits be used to label other proteins besides antibodies?
A:The principle of biotin labeling kits is that the ester bond of activated biotin reacts with the amino group on the target material to form a stable amide bond. Additionally, the N-terminus of proteins can be labeled. If a protein has primary amines, such as lysine, it can be labeled.
Q9:For protein labeling using the biotin labeling kit, is the reaction ratio 20:1?
A:The molecular weight of the protein and the number of lysines it carries should be considered. Generally, it is recommended to label at a concentration of 1–5 mg/mL. If the number of lysines is around 10–15, a ratio of 1:20 can be used. If there are more lysines, the ratio should be adjusted.
Q10:How to detect the efficiency of biotin-labeled antibodies?
A: The biotin labeling kits we offer have been optimized for the labeling ratio of numerous antibodies. When considering the recovery rate of the labeled antibodies, it is typically above 80%. The greater the quantity of labeled antibody, the higher the recovery rate. It's important to note that the calculation method for biotin labeling efficiency differs among manufacturers and should not be the sole criterion for evaluating the labeling effect.
Q11:How long is the shelf life of dissolved activated biotin/small molecule fluorescent dyes?
A:The dry powder of activated biotin/small molecule fluorescent dyes can be stably stored for over a year at -20°C before opening. However, after opening/dissolving, it should be sealed well, protected from light and moisture, and used within a week.
Q12:Is the protein labeled with biotin or small molecule dyes sterile, and how to sterilize or disinfect it?
A:The labeling process is usually carried out in an open area, so the labeled protein will not be completely sterile. Methods for sterilization or disinfection include:
For small volumes (e.g., 50–500 μL) of labeled protein, vacuum freeze-drying can be used for initial sterilization, or high-speed centrifugation can be used to precipitate bacteria and take the supernatant to remove most of the bacteria.
For larger volumes (greater than 1 ml) of labeled protein, a needle-type sterile filter can be used for filtration and sterilization.
Q13:How to detect and calculate the efficiency of protein labeling with biotin/small molecule dyes?
A:There are several ways to understand the calculation of labeling efficiency:
1)DOL: The average number of dyes labeled per protein. The higher the DOL, the higher the labeling efficiency.
2)The ratio of DOL to the feed ratio. For example, if the feed ratio of dye to protein is 10 and the final DOL is 5, then the labeling efficiency is 50%.
3)Antibody recovery rate: Measure the amount of antibody before and after labeling. The less antibody loss after labeling, the higher the recovery rate and labeling efficiency.
Q14:How long can labeled antibodies be stored at 4°C?
A:Labeled antibodies stored in our labeling preservation solution can be stably stored for more than half a year at 4°C. Antibodies labeled with small molecule dyes can be stably stored for over a year.
Q15:Are there any requirements for the size of the labeled antibodies or proteins?
A:The labeling kits currently available suggest labeling antibodies or proteins around 150 kDa. If customers need to label proteins of other sizes, we can customize and adjust the process. However, in general, the suggested protein size should not be less than 20 kDa.
Q16:Are there any antibodies that are not suitable for labeling?
A:Antibodies or proteins containing bovine serum albumin (BSA), gelatin, free amino acids or ammonium salts are not suitable for labeling.
Q17:How many fluorophores can generally bind to one antibody? Will different antibodies have different numbers of labeled fluorophores?
A: For the same fluorophore, under identical labeling conditions, the number of labeled fluorophores on various antibodies is relatively small, unless there are notable differences in the antibody source or buffer composition. Typically, an antibody can be labeled with 2 to 9 fluorophores. Once the Degree of Labeling (DOL) exceeds 5, the increase in brightness becomes insignificant, although the background value might rise. For fluorophores that are resistant to quenching, up to 9 can be attached. In exceptional cases, certain specialized antibodies paired with specific fluorophores can achieve more than 10 or as few as 2, still maintaining good fluorescence intensity.
Q18:How is the labeling efficiency DOL detected and calculated?
A:We measure the molar ratio of purified dye/antibody protein. There are also methods using SDS-PAGE electrophoresis or SEC purification columns for determination. The results from different methods may vary slightly, but generally, the differences are not significant.
Q19:Why is the fluorescence usually brighter after labeling with EnkiLife labeling kits?
A:The fluorescent dyes used in our labeling kits are selected from multiple fluorescent dye raw material suppliers and have undergone strict quality testing. Additionally, some dye raw materials have been chemically treated to maintain their stability.
Q20:What is the general ratio of small molecule fluorescent dyes/biotin to protein?
A:When the antibody concentration is 1 mg/ml, the molar ratio is approximately 1:20. There may be some differences for different fluorescent dyes/biotin, but it is generally between 10:1 and 30:1.
Q21:Is the principle of labeling antibodies with small molecule fluorescent dyes/biotin the same as that with large molecule protein fluorescent dyes, and what are the differences?
A: Small molecule fluorescent dyes and biotin utilize SE (succinimidyl ester NHS) to link with the -NH2 groups of antibodies, forming a structure akin to a peptide bond. This is the simplest method of connection. In contrast, large molecule protein fluorescent dyes employ the Maleimide active group to bind with thiol (-SH) groups. The advantage of this method is that it specifically labels the Fc fragment of the antibody, leaving the antigen unaffected.
Q22:Can I use the antibody labeling kit if my antibody contains glycerol?
A: It is best if the glycerol content is below 20%. If the glycerol content is higher, it can be reduced by using an ultrafiltration tube for multiple rounds of buffer exchange. However, this may affect the recovery rate of the antibody.
Q23:Can I use the antibody labeling kit if my antibody contains BSA?
A: For large molecule protein fluorescent dye labeling kits, a small amount of BSA (below 0.2%) is permissible. For small molecule fluorescent dye/biotin labeling kits, the presence of BSA can significantly impact the labeling results. We offer a BSA removal kit for antibodies, which can be used to remove BSA before labeling to achieve better results.
Q24:Why does your kit require so many ultrafiltration steps? I see that other manufacturers' labeling procedures are much simpler.
A: To ensure optimal labeling results, our labeling kits usually process the antibody raw material to eliminate impurities that could interfere with the labeling process. Following labeling, certain kits incorporate a purification step to further minimize the impact of unreacted dye on subsequent detection results, such as reducing background staining or for in vivo applications. If the customer's antibody lacks interfering factors and has the appropriate concentration, some steps can be simplified. The total time required for labeling is not markedly different from that of other manufacturers.
Q25: How should I choose a labeling kit if I need to label more than 10 mg of antibody?
A: Since large-scale and small-scale labeling processes differ, we currently do not provide a specific labeling kit for 10 mg of antibody. If the 10 mg of antibody is of the same type, you can opt for ten 1 mg kits or consider our labeling service. Should the 10 mg of antibody consist of various types, you can select based on the required fluorescent type and the labeling cost.
 | Hansun Hansun is a protein labeling expert at EnkiLife, proficient in immunology, labeling techniques, and flow cytometry, dedicated to precise protein labeling and empowering technological breakthroughs. Every protein deserves to be 'seen'. |