Cat. No. CLK-B105-10 CLK-B105-100 Amount 10 mg 100 mg For in vitro use only! Quality guaranteed for 12 months Store at -20 C 1. Description UV-Tracer TM Biotin Maleimide for biotinylation reactions of thiol (-SH)-containing proteins, e.g. IgGs (100-1000 µg in 500 µl), with a unique linker design that facilitates biotinylation and rapid quantification of the exact numbers of incorporated biotin molecules in a one - step procedure. Molecular Formula: C 54H 79N 70 15S Molecular Weight: 1098.31 g/mol Purity: > 90 % (HPLC) Solubility: DMSO, DMF, DCM Table of contents 1. Description 2. Protein requirements 3. Other important considerations 4. Biotinylation experiment 4.1 Additional material required 4.2 Preparation of protein 4.3 Equilibration of Spin Column 4.4 Buffer exchange of protein 4.5 Reduction of protein 4.6 Biotin labeling reaction 4.7 Removal of excess Biotin 5. Calculation of Labeling Degree (DOL) and protein concentration 6. Troubleshooting 7. Appendix A 8. Appendix B Figure 1 Chemical structure of UV-Tracer TM Maleimide. Red: UV-traceable benzophenone moiety. The linker contains a UV-traceable benzophenone moiety (Abs.: 350 nm) surrounded by two hydrophilic PEG spacer arms (Fig.1). The two hydrophilic PEG spacer arms enhance water solubility thereby reducing the tendency of some proteins to aggregate during biotinylation. The traceable chromophore permits rapid quantification of incorporated biotin molecules by a simple absorbance measurement at 280 nm and 350 nm. The resulting absorbance values are subsequently used to calculate the final protein concentration and the exact number of incorporated biotin molecules (Fig. 2). Figure 2 UV scan (230-450 nm) of Goat IgG (unlabeled control) (A), and UV-Tracer TM Biotin Maleimide labeled Goat IgG (B). Samples were scanned (1:4 dilution, c=0.425 mg/ml) in BupH (ph 6.5). The degree of labeling was determined to be 3.8 biotins/igg. Page 1 of 6 www.jenabioscience.com Last update: Jan 08, 2016
2. Protein requirements a) 100-1000 µg protein in a fixed volume of 500 µl (e.g. 0.2-2 mg/ml). b) The molecular weight needs to be known (e.g. 20-200 kda). c) Free thiol groups are required (e.g. cysteine). If they are absent, sulfhydryls can be added to proteins using N-succinimidyl S-acetylthioacetate (e.g. SATA available from ThermoScientific). d) The protein preparation needs to be highly purified and has to be free of reducing agents (e.g. β-me, DTT or TCEP) prior to labeling with maleimide esters. If they are present, these compounds must be removed (see 4.2b). e) Buffer exchange columns are designed to process 100-1000 µg of protein in a volume of 500 ± 50 µl. 3. Other important considerations a) Use the UV-Tracer TM Biotin Maleimide reagent immediately after reconstitution in DMSO. Although the maleimide group is more stable than other functional groups, it will hydrolyze thereby forming a nonreactive maleimic acid. Store the unused stock solution under moisture-free conditions (e.g. capped under an inert gas such as argon or nitrogen) at 4 C. Equilibrate reagent vial at room temperature before opening to avoid moisture condensation inside the vial. b) In every labeling reaction, the simplified protocol uses a fixed volume (10 µl) of UV Tracer TM Biotin Maleimide solution to label a fixed volume of protein (~ 525 µl). Consequently, the volume of anhydrous DMSO required to dissolve the UV Tracer TM Biotin Maleimide varies for each labeling reaction (see Appendix A, Part II for the calculation of the DMSO volume). c) The amount of biotin incorporated during the labeling reaction depends primarily on the number and availability of reduced thiols. Generally, a 10- to 20-fold molar excess of UV Tracer TM Biotin Maleimide solution is sufficient for most proteins. Over modification of the protein with biotin can affect both function and aqueous stability. 4. Biotinylation experiment 4.1 Additional material required: DMSO EDTA TCEP-HCL Reducing Agent thiol-free buffer (e.g. MOPS, Tris-HCL, PBS-buffer ph 6.5 or BupH Saline Buffer) Zeba TM Spin Columns UV-VIS Spectrophotometer Table Top Centrifuge (e.g. Eppendorf 5810) 1 l beaker stir bar with magnetic stir bar 15 ml conical tubes Ultrapure water (e.g. 18 MΩ-cm) 4.2 Preparation of protein a) If the protein is lyophilized (100-1000 µg), pure and free of exogenous thiols (e.g. DTT or β-me), resuspend in 500 µl PBS-buffer ph 6.5 to obtain a 0.2-2 mg/ml solution. Then proceed to the protein reduction step (4.5). b) If the protein is lyophilized and known to contain exogenous thiols (e.g. DTT, β-me) resuspend in 500 µl PBS-buffer ph 6.5 and then proceed with buffer exchange (4.3 and 4.4). Proceed afterwards to the protein reduction step (4.5). c) If the protein (100-1000 µg) is supplied in 500 µl of a suitable thiol-free buffer (e.g. MOPS, Tris-HCL or PBS) at a concentration range from 0.2-2 mg/ml, proceed to the protein reduction step (4.5). d) If the protein is supplied in a buffer known to contain exogenous thiols (e.g. DTT, β-me) proceed with buffer exchange (4.3 and 4.4). Proceed afterwards to the protein reduction step (4.5). 4.3 Equilibration of Spin Column a) Twist off the column s bottom closure and loosen the cap. Place each column into a clean 15 ml conical tube. b) Centrifuge the column at 1,000 x g for 2 minutes to remove storage solution. Place a pen mark on the side of the column where the compacted resin is slanted upward. Place the column in the centrifuge with the Page 2 of 6 www.jenabioscience.com Last update: Jan 08, 2016
mark facing away from the center of the rotor in all subsequent centrifugation steps. Please note: The resin will appear white in color and compacted after centrifugation. c) Add 1 ml PBS-buffer ph 6.5 to the top of each spin column and remove the cap. d) Centrifuge at 1000 x g for 2 minutes to remove buffer. e) Repeat steps 3 and 4 two additional times, discarding buffer from the collection tube after each spin. f) Transfer the equilibrated spin column (resin appears white and dry) into a clean 15 ml conical tube and proceed immediately with the buffer exchange of the protein. 4.4 Buffer exchange of protein a) Slowly apply 500 µl protein solution to the center of the equilibrated spin column (4.3). b) Centrifuge at 1,000 x g for 2 minutes. c) The eluate at the bottom of the 15 ml collection tube contains the buffer exchanged protein. Discard the used spin column. 4.5 Reduction of protein Prior to biotinylation, proteins containing disulfide bonds must be reduced with TCEP to insure proper labeling. a) Add 500 µl ultrapure water to a vial containing 15 mg TCEP-HCl (105 mm) and vortex to completely dissolve the crystals. b) Add 5 µl 500 mm EDTA to 500 µl protein solution (0.2-2 mg/ml) and pipette the mixture several times up and down. c) Add 25 µl dissolved TCEP (105 mm) to the protein solution and pipette several times up and down. d) Incubate the reaction for 30 minutes at room temperature. e) Prepare a buffer exchange spin column as described in section 4.3. f) Buffer exchange TCEP reduced protein as described in section 4.4. g) Immediately after buffer exchange, add 5 µl 500 mm EDTA to the protein solution and pipette the solution several times up and down. 4.6 Biotin labeling reaction a) Select the amount of excess UV-Tracer TM Biotin Maleimide for the labeling reaction (refer to Appendix A, Part I as a reference guide). b) Determine the required volume of DMSO to dissolve the required amount of UV-Tracer TM Biotin Maleimid (refer to Appendix A, Part II). c) Add the required volume of DMSO to the UV- Tracer TM Biotin Maleimide and vortex vigorously for 2 minutes until the reagent is fully dissolved. d) Add 10 µl UV-Tracer TM Biotin Maleimide solution to the reduced protein solution (~ 525 µl) and pipette the mixture several times up and down. e) Incubate the reaction mixture for 1 hour at room temperature. 4.7 Removal of excess Biotin a) Prepare a buffer exchange spin column as described in section 4.3. b) Buffer exchange the biotinylated protein as described in section 4.4 thereby removing excess (= unbound) UV-Tracer TM Biotin Maleimide. You may use the buffer of your choice instead of BupH buffer, ph 6.5. 5. Calculation of Labeling Degree (DOL) and protein concentration a) Measure the conjugate absorbance at 280 nm and 350 nm with a UV-VIS-Spectrophotometer. Please note: Concentrated protein solutions (e.g. 2 mg/ml) will require dilution prior to absorbance measurements. A micro-volume spectrophotometer can be used on small aliquots (1-2 µl) without dilution (e.g. Nanodrop ). Page 3 of 6 www.jenabioscience.com Last update: Jan 08, 2016
b) Calculate the degree of labeling (DOL) and protein concentration with the calculations found in Appendix B, Part I. Page 4 of 6 www.jenabioscience.com Last update: Jan 08, 2016
6. Troubleshooting Problem 1: Poor or lower biotinylation efficiency than expected Possible Cause Incorrect protein concentration and/or possible contaminants in protein sample. Maleimide-ester hydrolyzed Solution Buffer exchange the protein into BupH buffer (ph 6.5) using spin columns provided and confirm the concentration of the protein prior to labeling. Store UV-Tracer TM Biotin Maleimide reagent at -20 o C. Allow product to equilibrate to room temperature before opening. Avoid buffers that may contain free thiols (β-me or DTT). Buffer exchange proteins before labeling whenever possible. Appendix A Part I Determination of the required UV-Tracer TM Biotin Maleimide amount a) Select the desired molar excess of UV-Tracer TM Biotin Maleimide (refer to Table 1 as a reference for the selection process). Typical labeling reactions use 5x to 20x reagent molar excess depending on the initial protein concentration and the number of available thiols. Please note: Over modification of antibodies or other proteins with biotin can affect their function and stability. Table 1 Typical labeling efficiencies achieved for goat IgG (possess 32 cystein residues). DOL: Degree of Labelig. Protein has few or no available thiol residues Low A350 absorbance of the biotinylated conjugate Check primary structure of protein for the presence of cysteine residues on NCBI protein database. Some proteins may require modification with SATA (or similar reagent) to introduce thiol functional groups. Check spectrophotometer lamp for proper functioning. b) Calculate millimole (mmol) UV-Tracer TM Biotin- Maleimide (MW: 1098.31 g/mol) required to label a protein with a desired molar excess: Problem 2: Low conjugate yield Possible Cause Protein may have aggregated/ precipitated during biotinylation Solution Use appropriate relative centrifugal force (e.g. 1000 x g) and the recommended spin time to buffer exchange the protein. Although rare, some proteins become unstable in aqueous solution on biotinylation and cannot be labeled. Nb: molecular excess of UV-Tracer TM Biotin Maleimide Cp: protein concentration [mg/ml] Vp: volume of protein sample [ml] (fixed: 0.5 ml) MWp: molecular weight of protein [Dalton] Example: 0.000133 mmol of UV-Tracer TM Biotin Maleimid are required to label 0.5 ml of a 2 mg/ml IgG solution (MW p=150 kda) with a 20-fold molar excess. This equals 0.146 mg of UV-Tracer TM Biotin Maleimid. Page 5 of 6 www.jenabioscience.com Last update: Jan 08, 2016
Part II Determination of the DMSO volume (µl) required to dissolve a desired amount of UV-Tracer TM Biotin Maleimide (Part I) a) Calculate microliters anhydrous DMSO required to dissolve UV-Tracer TM Biotin Maleimide reagent: ()!"#$%&'(% () *) 0.0364 mmol UV-Tracer TM Biotin (vial) = 0.003642 mmol (=4 mg) VUV-Tracer : Required volume of UV-Tracer TM solution = 10 µl Example: Dissolve 1 vial of UV-Tracer TM Biotin Maleimide with 273.8 µl anhydrous DMSO and mix 10 µl of this solution with the protein to achieve a 20x molar excess of UV-Tracer TM Biotin Maleimide in the final solution (4.6). Please note: Each vial of UV-Tracer TM Biotin Maleimide can accommodate 1,600 µl DMSO. If the required volume of DMSO is greater than this nominal volume, transfer the dissolved reagent to a larger vial and add DMSO to achieve the required volume. Appendix B Part I a) Calculation of the Degree of Labeling (DOL) according to the law of Lambert-Beer: (0) 123425[μ] (?) @%34(25[μ] (D) 123425 @%34(25 9 :;< = :;< > 9 AB<C = AB< > d = layer thickness of cuvette (e.g. 1 cm) A350 = UV-Tracer TM Biotin absorbance at 350 nm ε350 = molar extinction coefficient of UV-Tracer TM Biotin =19474 M -1 cm -1 A280 = protein conjugate absorbance at 280 nm A280c = corrected protein conjugate absorbance at 280 nm = A280 (A350 x 0,4475)) ε280 = molar extinction coefficient of protein b) Calculate conjugate protein concentration (mg/ml) (E) @%34(25 F G H %34(25 [μ] cprotein [µm] = determined with Eq. (2) MWp = molecular weight of protein [Dalton] >I J Example: A Goat IgG antibody (MW p = 150 kda) was labeled using a 20x molar excess of UV-Tracer TM Biotin Maleimide. The conjugates absorbance at 280 nm and 350 nm were determined with a quartz cuvette (d= 1 cm): A 280 = 0.67 (1:4 dilution) A 350 = 0.21 (1:4 dilution) ε280 (IgG) = 204000 M -1 cm -1 Results according to (1) (4): (1) c Biotin = 10.78 µm (2) c Protein = 2.82 µm (3) DOL = 3.82 (4) c Protein = 1.7 mg/ml Page 6 of 6 www.jenabioscience.com Last update: Jan 08, 2016