| Description | Aladdin's Anti-V5 Affinity Gel, also known as Anti-V5 IP Gel, Anti-V5 immunoprecipitation gel or Anti-V5 agarose gel, is agarose gel covalently coupled with high-quality mouse monoclonal antibody that recognizes the V5 tag sequence (GKPIPNPLLGLDST). This product can specifically bind V5-tagged Aladdin's Anti-V5 Affinity Gel, also known as Anti-V5 IP Gel, Anti-V5 immunoprecipitation gel or Anti-V5 agarose gel, is agarose gel covalently coupled with high-quality mouse monoclonal antibody that recognizes the V5 tag sequence (GKPIPNPLLGLDST). This product can specifically bind V5-tagged proteins expressed in animals, plants, and microorganisms, and can be used for immunoprecipitation (IP) and purification of V5-tag fusion proteins or their protein complexes.V5-tag is a peptide consisting of 14 amino acids (GKPIPNPLLGLDST) isolated from monkey parainfluenza virus V5, which can be fused either to the N-terminus or the C-terminus of a target protein to facilitate protein purification and immunoassays. V5-tag usually does not interact with the target protein, and in most cases does not affect the function of the target protein; With Aladdin's V5 antibodies, Anti-V5 magnetic beads, or Anti-V5 affinity gel, V5-tagged proteins can be used for examination of protein expression level and subcellular localization, protein purification, and immunoassays.This product has high binding capacity and strong specificityPrecautions：This product must be fully resuspended by inverting the tube several times prior to use.This product contains a small amount of preservative, which does not affect routine IP assays and protein purification. But for special experiments that might be interfered by the preservative, the gel should be washed 3 times with appropriate solutions such as TBS prior to use.For immunoprecipitation assays, it is recommended to include both positive and negative controls. Protein samples should be purified as soon as possible after collection and should always be placed at 4℃ or on ice to minimize protein degradation or denaturation. Protein degradation can be inhibited by adding appropriate protease inhibitors, such as Protease Inhibitor Cocktail for General Use, Protease and Phosphatase Inhibitor Cocktail for General Use (MS-safe, 50X), Protease Inhibitor Cocktail for Mammalian Cell and Tissue Extracts, Protease and Phosphatase Inhibitor Cocktail for Mammalian Cell and Tissue Extracts, etc.High concentrations of DTT, mercaptoethanol, guanidine hydrochloride, etc. may have a certain effect on the binding of this product to V5-tagged proteins, but Cell lysis buffer for Western and IP, RIPA Lysis Buffer or NP-40 Lysis Buffer are applicable to this product. For the main features and differences of various lysis buffers produced by and the selection of lysis buffers, please refer to the website at http://www.aladdin-e.com/support/lysis-buffer.htm.If the insoluble substance in protein samples can not be removed completely by centrifugation, samples can be filtered with a 0.45µm filter before performing the assay.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.Instructions for Use：1.Preparation of protein samplesa.Lysis cells or tissues with appropriate lysis buffer, such as ’s Cell Lysis Buffer for Western and IP. Under certain circumstances, ’s RIPA Strong Lysis Buffer, RIPA Medium Lysis Buffer, or RIPA Weak Lysis Buffer can also be used. Other lysis buffers with a pH6-8 can also be used.b.After lysis and centrifuge, keep the supernatant at 4℃ or on ice for subsequent use. We recommend performing the subsequent procedures on the same day. Otherwise, make aliquots and store them at -80℃ for future use.2.Preparation of Anti-V5 Affinity GelAs the Anti-V5 Affinity Gel is stored in a protective solution containing 50% glycerol, it needs to be washed properly before adding protein samples.a.Gently resuspend the Anti-V5 Affinity Gel to be homogeneous. Transfer 20µl of gel suspension per 100µl of protein sample into a clean centrifuge tube. Note: It is easier to aspirate the gel suspension using a large aperture pipette tip (e.g., by cutting off part of the tip with scissors).b.Add 1X TBS to a final volume of 0.5ml, and gently resuspend the Anti-V5 Affinity Gel by pipetting or vortex. Centrifuge at 6000×g for 30 seconds at 4℃, remove the supernatant carefully without disturbing the gel. Repeat this step twice.c.Resuspend the Anti-V5 Affinity Gel with 1X TBS equal to the initial volume of gel suspension (e.g., if 20µl of gel suspension is taken, add 20µl of 1X TBS).3.Protein binding1)Add 20µl of washed Anti-V5 Affinity Gel per 100µl of protein sample, mix well, and incubate at 4℃ for 1-2 hours or overnight with gentle shaking on a rotary mixer.2)After incubation, centrifuge at 6000×g for 30sec at 4℃ and remove the supernatant carefully without disturbing the gel. Note: A portion of supernatant can be reserved in a clean microfuge tube for examination of the binding results.3)Gently resuspend the Anti-V5 Affinity Gel in 500µl of 1X TBS and place in an ice bath on a shaker for 5 minutes. Centrifuge at 6000×g for 30 seconds at 4℃ and carefully remove the supernatant. Repeat the wash thrice, then keep the sample on ice for elution. 4.ElutionBased on the features of the target protein or downstream applications, one of the following three elution methods can be used.1)Competitive elution with the V5 peptide. This is a non-denaturing elution method with a high elution efficiency, and the eluted proteins do not contain the light and heavy chains of V5 antibody.a.Preparation of V5 peptide elution buffer: Dissolve the V5 Peptide (, P9813) in 1X TBS buffer to a final concentration of 150µg/ml, or dilute the 5mg/ml V5 peptide solution with 1X TBS buffer to a final concentration of 150µg/ml. b.For every 20µl of initial gel volume, add 100µl of V5 Peptide elution buffer (150µg/ml). Resuspend the gel gently and incubate for 30-60 minutes at room temperature on a rotary mixer, or 1-2 hours at 4℃. To improve the elution efficiency, increase the incubation time or repeat the elution. c.After incubation, centrifuge at 6000×g for 30 seconds at 4℃, and transfer the supernatant to a new microfuge tube. The supernatant contains the V5-tagged protein and its protein complex.d.Store the supernatant at 4℃ for immediate use, or -20℃/ -80℃ for long-term storage. 2)Acidic elution: This method is non-denaturing, relatively fast and efficient. The eluted proteins also retain their original biological activity, which facilitates subsequent analysis and detection.a.Preparation of acidic elution buffer (0.1M Glycine-HCl, pH3.0) and neutralization buffer (0.5M Tris-HCl, pH7.4, 1.5M NaCl). b.For every 20µl of initial gel volume, add 100µl of acidic elution buffer. Resuspend the agarose gently and incubate for 5 minutes at room temperature on a rotary mixer.Note: The incubation time should not exceed 15 minutes.c.After incubation, centrifuge at 6000×g for 30 seconds at 4℃, and transfer the supernatant to a new microfuge tube. Immediately add 10µl of neutralization buffer and mix well. d.To achieve the maximum elution efficiency, repeat steps b-c and combine the supernatant containing the V5-tagged protein and its protein complex. e.Store the supernatant at 4℃ for or immediate use, or -20℃/-80℃ for long-term storage. Note 1: The elution efficiency of the acidic elution method might be lower than the other two elution methods.Note 2: The elution efficiency of the acidic elution method depends on characteristics of the target protein. To obtain a higher elution efficiency, the pH of the acidic elution buffer can be optimized from 2.5 to 3.1. The pH or amount of neutralization buffer needed to neutralize the eluates should also be adjusted appropriately.3)Elution with the 1X SDS-PAGE loading buffer: This method is a denaturation method, and the obtained proteins are suitable for SDS-PAGE electrophoresis or WB blot analysis.a.Preparation of SDS-PAGE loading buffer: We recommend using ’s SDS-PAGE Sample Loading Buffer (2X). Usually, the SDS-PAGE protein loading buffer contains a reducing agent such as DTT, and the eluted protein sample will contain the light chain and heavy chain of the V5 antibody.b.For every 20µl of initial gel volume, add 20µl of 2X SDS-PAGE sample loading buffer, mix well and heat at 95℃ for 5 minutes. c.Centrifuge at 6000×g or 4℃ for 30 seconds. Take the supernatant for SDS-PAGE electrophoresis or Western blot analysisNote: The Agarose cannot be reused because SDS in the loading buffer destroys V5 antibodies.FAQ: Problem Possible Causes Solution Large amount of tagged protein found in the flow through. Binding time is not enough. If using batch method, increase the binding time experimentally; If using column method, use a lower flow rate when loading samples. Column is overloaded. Reduce the amount of the sample added to the gel or increase the amount of gel. Tag is not accessible to gel. Expose the epitope tag by adding low amount of denaturant to the protein extract (dialysis may be needed before applying onto gel), or fuse thetag to the other terminus of the target protein. Gel has not been regenerated since last purification. Perform gel regeneration procedure prior to binding. Reagent compatibility problem. Dialyze the sample against TBS before purification procedure. The target protein has been degraded. 1.Prepare fresh lysates. Avoid using frozen lysates.2.Perform purification at lower temperature, such as 4℃.3.Use appropriate protease inhibitors in the lysate or increase their concentrations to prevent degradation of the fusion protein. Very few or notagged protein exists in the eluate. Protein is not completely eluted. Change elution methods. No target protein expressed. Make sure the protein of interest contains the tag by Western blot or dot blot analyses. Very low protein expression level. 1.Use larger volume of cell lysate.2.Optimize expression conditions to raise the protein expression level. Washes are too stringent. 1.Reduce the number of washes.2.Avoid adding high concentrations of NaCl to the mixture.3.Use solutions that contain less or no detergent Incubation times are inadequate. Increase the incubation times with the affinity gel (from several hours to overnight). Interfering substance is present in sample. 1.Lysates containing high concentrations of DTT, 2-mercaptoethanol, or other reducing agents may destroy antibody function, and must be avoided.2.Excessive detergent concentrations may interfere with the antibody-antigen interaction. Detergent levels in buffers may be reduced by dilution. Detection system is inadequate. If Western blot detection is used:1.Check primary and secondary antibodies using proper controls to confirm binding and reactivity.2.Verify that the transfer was adequate by staining the membrane with Ponceau S.3.Use fresh detection substrate or try a different detection system. Multiple protein bands found in the eluate. The protein is not stable at room temperature. Purify the target protein at lower temperature, such as 4℃. Protein degradation due to proteases activity during purification process. Add protease inhibitors to cell lysate. Non-specific binding. 1.Prepare cell lysate again.2.Add additional wash steps. Background is too high. Proteins bind nonspecifically to the monoclonal antibody, the gel beads, or the microcentrifuge tubes. 1.Pre-clear lysate with Mouse IgG-Agarose to remove nonspecific binding proteins.2.After suspending beads for the final wash, transfer entire sample to a clean microcentrifuge tube before centrifugation. Washes are insufficient. 1.Increase the number of washes.2.Prolong duration of the washes, incubating each wash for at least 15 minutes.3.Increase the salt and/or detergent concentrations in the wash solutions.4.Centrifuge at lower speed to avoid nonspecific trapping of denatured proteins from the lysate during the initial centrifugation of the affinity gel complexes... Read More | Inquire | 2x Taq MasterMix is a premixed system composed of Taq DNA Polymerase, Mg2+, dNTPs, PCR stabilizers, and enhancers. The pre prepared PCR mixture makes the operation simpler and faster, and can minimize human error and contamination to the greatest extent possible. The original MasterMix formula 2x Taq MasterMix is a premixed system composed of Taq DNA Polymerase, Mg2+, dNTPs, PCR stabilizers, and enhancers. The pre prepared PCR mixture makes the operation simpler and faster, and can minimize human error and contamination to the greatest extent possible. The original MasterMix formula results in high yield, strong repeatability, and good stability of amplified products. This product does not contain dyes. After the PCR program is completed, an appropriate amount of sample buffer can be added as needed for electrophoresis operation. The amplified PCR product has an "A" base attached to the 3 'end, making it suitable for direct use in T/A cloning. Mainly suitable for PCR amplification of DNA, DNA sequencing and other experiments.Quality control: T665627Component5mlStorageT665627A2×Taq MasterMix5×1ml-20℃. Avoid freeze/thaw cycle.T665627BddH₂O5×1ml-20℃. Avoid freeze/thaw cycle.Notes: 2×Taq MasterMix contains Taq DNA Polymerase, 3mM MgCl2 and 400µM each dNTP After testing, there was no exogenous nuclease activity; PCR method for detecting residual DNA without host; Can effectively amplify single copy genes from multiple genomes.Usage:The following is an example of a PCR reaction system and reaction conditions for amplifying a 1 kb fragment using human genomic DNA as a template. In practical operation, corresponding improvements and optimizations should be made based on the template, primer structure, and target fragment size.1. PCR reaction systemReagent50 µlReaction systemFinal concentration2×Taq MasterMix25 µl1×Forward Primer，10 µM2 µl0.4 µMReverse Primer，10 µM2 µl0.4 µMTemplate DNA<0.5 µg<0.5 µg/50 µlddH2Oup to 50 µl/Attention: The primer concentration should be between 0.1 and 1.0 as the final concentration µ M serves as a reference for setting the range. In the case of low amplification efficiency, the concentration of primers can be increased; When non-specific reactions occur, the primer concentration can be reduced to optimize the reaction system.2. PCR reaction conditionsStepTemperatureTime/Pre denaturation95℃2 min/Denaturation94℃30 s25-35 cyclesAnneal55-65℃30 s25-35 cyclesExtend72℃30 s25-35 cyclesFinally extended72℃2 min/Attention:1) In general experiments, if the annealing temperature is 5 ℃ lower than the melting temperature Tm of the amplification primer, and the ideal amplification efficiency cannot be achieved, the annealing temperature should be appropriately reduced; When non-specific reactions occur, increase the annealing temperature to optimize the reaction conditions.2) The extension time should be set according to the size of the amplified fragment. The amplification efficiency of Taq DNA Polymerase in this product is 2 kb/min.3) The number of cycles can be set based on the downstream application of the amplification product. If the number of cycles is too small, the amplification amount is insufficient; If there are too many cycles, the probability of mismatches will increase, and non-specific backgrounds will be severe. So, while ensuring product yield, the number of cycles should be minimized as much as possible... Read More | Purity＞97% by SDS-PAGE and HPLC analyses.FunctionPigment epithelium-derived factor (PEDF) is encoded by the SERPINF1 gene in humans and found in verebrates. It is a secreted phosphoglycoprotein that belongs to the clade F subfamily, serpin superfamily of proteinase inhibitors. The PEDF is a Purity＞97% by SDS-PAGE and HPLC analyses.FunctionPigment epithelium-derived factor (PEDF) is encoded by the SERPINF1 gene in humans and found in verebrates. It is a secreted phosphoglycoprotein that belongs to the clade F subfamily, serpin superfamily of proteinase inhibitors. The PEDF is a noninhibitory serpin with neurotrophic, anti-angiogenic, and anti-tumorigenic properties. It is synthesized as a 418 a.a. about 50kDa precursor that contains a 19 a.a. signal sequence and a 399 a.a. mature region that shows a pyroglutamate at Gln20. Like other serpins, it contains three β-sheets, 810 α-helices, and a C-terminal RCL (reactive center loop). Unlike other serpins with Ser protease inhibiting activity. PEDF has functions of inducing extensive neuronal differentiation in retinoblastoma cells, inhibiting of angiogenesis. As it does not undergo the S (stressed) to R (relaxed) conformational transition characteristic of active serpins, it exhibits no serine protease inhibitory activity. PEDF is researched as a therapeutic candidate for treatment of such conditions as choroidal neovascularization, heart disease, and cancer... Read More | Purity:>90%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description：SOD2 is part of the iron/manganese superoxide dismutase family. It encodes a mitochondrial protein that forms a homotetramer and binds one manganese ion per subunit. SOD2 binds to the superoxide byproducts Purity:>90%, by SDS-PAGE visualized with Coomassie® Blue Staining.Description：SOD2 is part of the iron/manganese superoxide dismutase family. It encodes a mitochondrial protein that forms a homotetramer and binds one manganese ion per subunit. SOD2 binds to the superoxide byproducts of oxidative phosphorylation and converts them to hydrogen peroxide and diatomic oxygen. Mutations in SOD2 gene have been associated with idiopathic cardiomyopathy (IDC), premature aging, sporadic motor neuron disease, and cancer. SOD2 destroys radicals which are usually produced within the cells and which are toxic to biological systems... Read More |