| Description | This kit innovatively adopts a column-based purification method to rapidly, gently, and efficiently lyse animal tissues or cells for total protein extraction. It provides both denaturing and native lysis buffers, allowing users to select the appropriate option based on downstream application This kit innovatively adopts a column-based purification method to rapidly, gently, and efficiently lyse animal tissues or cells for total protein extraction. It provides both denaturing and native lysis buffers, allowing users to select the appropriate option based on downstream application requirements. The entire extraction process takes only 1–8 minutes. Thanks to the column purification technology, it can process sample-lysis buffer mixtures as small as 20 µL and up to 500 µL, yielding protein solutions with concentrations of 2–8 mg/mL while effectively preventing protein loss. The extracted proteins can be quantified using the BCA method (Cat. No.: R1491648/B665595). C1491689 Component50TStorageC1491689ADenaturing Lysis Buffer25 mL2-8℃C1491689BNative Lysis Buffer25 mL2-8℃C1491689CProtease Inhibitor Cocktail500 µL-20℃C1491689DPurification Columns50 unitsRT.C1491689ECollection Tubes50 unitsRT.C1491689FPlastic Grinding Pestles4 unitsRT.Key Features1.Simple and rapid operation: Denatured total proteins can be obtained in as little as 1 minute.2.No protein loss: Efficiently extracts DNA-binding proteins by disrupting DNA duplexes.3.Small sample volume, high yield: Processes mixtures as small as 20 µL, yielding protein concentrations of 2–8 mg/mL.4.Versatile applications: Includes two lysis buffers for extracting both denatured and native proteins.Protocol1、Extraction of Denatured Total Protein1.Pre-chill the purification column and collection tube on ice.2.Sample processing: Add protease inhibitor cocktail to the denaturing lysis buffer at a 1:100 ratio shortly before use.2.1 Adherent cells:Wash cells with pre-chilled 1× PBS and aspirate the supernatant.Add the volume of denaturing lysis buffer specified in the appendix table to cover the culture surface, and pipette to mix.2.2 Suspension cells:Collect cells by low-speed centrifugation.Wash with pre-chilled 1× PBS, vortex, and centrifuge at 3,000 rpm for 2–3 minutes.Resuspend the cell pellet in PBS equal to the pellet volume.Add the specified volume of denaturing lysis buffer and vortex to lyse.Note: Partial incomplete lysis does not affect protein extraction. If the lysate is too viscous, directly transfer it to the purification column.2.3 Tissue samples:Place 15–20 mg of tissue on the purification column.Grind 50–60 times with a plastic pestle.Add 200 µL denaturing lysis buffer and grind another 30–60 times.Adjust lysis buffer volume proportionally for larger or smaller samples.Note: Reusable plastic pestles should be thoroughly rinsed with distilled water and dried.3.Centrifugation:3.1 Adherent or suspension cells: Transfer the lysate to the pre-chilled purification column and centrifuge at 14,000–16,000 rpm for 30 seconds.3.2 Tissue samples: Incubate the column at room temperature for 1–2 minutes, then centrifuge at 14,000–16,000 rpm for 1–2 minutes.4.Immediately place the collection tube on ice and discard the purification column. Denatured total protein extraction is complete.2、Extraction of Native Total Protein1.Pre-chill the native lysis buffer, purification column, and collection tube on ice.2.Sample processing: Add protease inhibitor cocktail to the native lysis buffer at a 1:100 ratio shortly before use.2.1 Adherent cells:Wash cells with pre-chilled 1× PBS and aspirate the supernatant.Add the specified volume of native lysis buffer and incubate on ice for 3–5 minutes. Pipette to mix.2.2 Suspension cells:Collect, wash, and resuspend cells as described in section I.Add native lysis buffer, vortex for 15 seconds, incubate on ice for 3–5 minutes, and vortex again for 10 seconds.2.3 Tissue samples:Grind tissue as described in section I, using native lysis buffer.3.Centrifugation:3.1 Adherent or suspension cells: Centrifuge at 14,000–16,000 rpm for 30 seconds.3.2 Tissue samples: Incubate on ice for 5 minutes (open lid), then close the lid and centrifuge at 4°C and 14,000–16,000 rpm for 1–2 minutes.4.Immediately place the collection tube on ice and discard the purification column. Native total protein extraction is complete. Cell Count (×10⁶)Lysis Buffer Volume (µL)0.3200.550110022003500 Appendix: Cell Number vs. Lysis Buffer VolumePrecautionsHigh viscosity of the lysate is normal when using this kit.For safety, wear a lab coat and disposable gloves during operation.For research use only... Read More | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Product Characteristics Effect Diluents, Animal-free are effective buffers free of any animal components. They can be used for the dilution of serum, plasma, blood, stool or urine samples, as well as the dilution of primary and secondary antibodies. Effect Diluents, Animal-free efficiently minimize Product Characteristics Effect Diluents, Animal-free are effective buffers free of any animal components. They can be used for the dilution of serum, plasma, blood, stool or urine samples, as well as the dilution of primary and secondary antibodies. Effect Diluents, Animal-free efficiently minimize matrix effects, cross-reactions and unspecific binding in immunoassays like ELISA, Western blotting, Immunohistochemistry, protein arrays and immuno-PCR.The Effect Diluents, Animal-free are used alternatively to the standard sample or antibody dilution buffers: In ELISA for the dilution of specimen and detection antibodies. In Western Blotting for the dilution of primary and secondary antibodies. In Protein arrays for the dilution of specimen and detection antibodies. In immuno-PCR as a washing buffer.Three versions of the diluent are offered: Low, Medium and High for optimal discrimination between specific and unspecific reaction and for minimizing strong interference effects e.g., by RF (rheumatoid factors), HAMAs (human-a-mouse Abs) or by endogenous components that bind and mask the analyte.Composition & Properties The Effect Diluents, Animal free contain no animal components and are free of phosphates.Working Procedure 1.Mix thoroughly prior to use. 2.Dilution recommendations a.Dilute antibodies according to the instruction of the antibody b.Dilution of the specimen is recommended at 1:2 or higherTips & TricksEffect Diluents must not be considered as blocking buffers. Recommended blocking buffers are: Synthetic Blocking Buffer, ELISA (cat. no. S494401), Synthetic Blocking Buffer, Blotting (cat. no. S494457) and WellChampion (cat. no. W494467) for plate blocking and stabilization (preparation of pre-coated plates). Complex sample matrices, such as serum and plasma, may contain interfering factors that affect the ability of the assay to accurately quantify the target analyte. Strong interferences are often caused by RFs and HAMAs. This matrix effect can cause high background in the negative control or false negatives in the sample measurement. To reduce this effect the samples can be diluted in the Effect Diluents, Animalfree.Handling & Storage Store solution 2-8°C or -15 to -30°C (tolerates freezing and thawing cycles)... Read More | Product content R669871Component50 TStorageR669871ADNase I1000 U-20℃. Avoid freeze/thaw cycle.R669871B10×Reaction Buffer1mL-20℃. Avoid freeze/thaw cycle. R669871CBuffer DS30 mLRTR669871DBuffer GTL15 mLRTR669871EBuffer GL25 mLRTR669871FProteinase K12.5 mgRTR669871GProteinase K Product content R669871Component50 TStorageR669871ADNase I1000 U-20℃. Avoid freeze/thaw cycle.R669871B10×Reaction Buffer1mL-20℃. Avoid freeze/thaw cycle. R669871CBuffer DS30 mLRTR669871DBuffer GTL15 mLRTR669871EBuffer GL25 mLRTR669871FProteinase K12.5 mgRTR669871GProteinase K Storage Buffer1.25 mLRTR669871HBuffer RW140 mLRTR669871IBuffer RW2 (concentrate)11 mLRTR669871JRNase-Free Water10 mLRTR669871KSpin Columns RS with Collection Tubes50 setsRTR669871LRNase-Free Centrifuge Tubes (1.5 mL)50 EART Product IntroductionThis kit is suitable for effectively purifying total RNA from formalin fixed and paraffin embedded tissues. Suitable for extracting total RNA with improved purity from paraffin embedded tissues or sections less than 30mg. This kit does not require the use of phenol/chloroform extraction or isopropanol precipitation, and can complete the extraction of multiple samples within one hour. This product uses specially optimized lysis solution and protease K to release RNA from formalin fixed or tissue slice samples without overnight operation; After digestion, the sample is incubated at a higher temperature to remove the inhibitory effect caused by formalin cross-linking, effectively releasing RNA from tissue slices and avoiding endangering RNA integrity; The optimized buffer system allows RNA in the lysis solution to specifically bind to the silica gel adsorption membrane, while other pollutants can flow through the membrane; It can be effectively removed through rinsing steps, and the washed RNA can be directly used for experiments such as RT-PCR, Real Time PCR, and Western blot analysis.Self prepared reagents: anhydrous ethanol (newly opened or dedicated for RNA extraction), 10mM PBS (pH 7.4).Preparation and important precautions before the experiment1. Add 0.625ml Protein K Storage Buffer to Protein K to dissolve it and store at -20 ℃. The prepared Protein K should not be left at room temperature for a long time to avoid repeated freeze-thaw cycles, which may affect its activity.2. To prevent RNase pollution, attention should be paid to the following aspects:1) Use RNase free plastic products and gun heads to avoid cross contamination.2) Glassware should be dry baked at a high temperature of 180 ℃ for 4 hours before use, while plastic containers can be soaked in 0.5M NaOH for 10 minutes, thoroughly rinsed with water, and then sterilized under high pressure.3) Prepare the solution using water without RNase.4) Operators should wear disposable masks and gloves, and change gloves frequently during the experiment.3. After obtaining the sample, it should be fixed in 4% -10% formalin as soon as possible, with a suitable fixation time of 14-24 hours. Excessive time can lead to RNA breakage and affect downstream experiments.4. Ensure that the sample before embedding is thoroughly dehydrated, as residual formalin will inhibit the action of Protein K.5. Before the first use, anhydrous ethanol should be added to Buffer RW2 according to the instructions on the reagent bottle label.Before use, please check if there is any crystallization or precipitation in Buffer GTL, Buffer GL, and Buffer DS. If there is any crystallization or precipitation, please dissolve Buffer GTL, Buffer GL, and Buffer DS again in a 56 ℃ water bath.Operation steps1. Sample processing1a. Paraffin embedded sample: Use a surgical knife to trim off excess paraffin from the tissue block, expose the tissue, and cut into 5-10 µ m thin slices.Attention: If the surface of the sample has already been exposed to air, please discard 2-3 pieces that come into contact with the air and do not use them.1b. Samples in fixed solutions such as formalin: Take approximately 20mg of the sample, cut it into small pieces, place it in a centrifuge tube, and add 500 µ 10mM PBS (PH7.4), vortex oscillation, centrifugation at 12000 rpm (~13400 × g) for 1 minute, discard the supernatant, repeat 3 times, and proceed directly to step 3.2. Choose option A or option B to remove paraffinOption AA1. Take approximately 1 × 1cm2 of slices (4-5 slices in total) and place them in a centrifuge tube (prepared by oneself), then add 500 slices µ L Buffer DS, vortex oscillation for 10 seconds. Incubate at 56 ° C for 3 minutes.Centrifuge at A2.12000 rpm for 2 minutes, be careful to discard the supernatant and avoid attracting sediment.Option BB1. Take approximately 4-5 slices of approximately 1 × 1 cm2 and place them in a centrifuge tube (self prepared). Add 1ml of xylene, cover the tube tightly, and vortex for 10 seconds.B2.Centrifuge at 12000 rpm for 2 minutes, be careful to remove the supernatant and avoid removing sediment.B3. Add 1ml of anhydrous ethanol, vortex and shake well. Centrifuge at 12000 rpm for 2 minutes, discard the supernatant, and be careful not to absorb or discard the sediment.B4. Open the tube cover and incubate at room temperature or up to 37 ° C for 10 minutes until there is no ethanol residue.3. Add 150µ L Buffer GTL, resuspended precipitation; Join 10µl Protein K, vortex oscillation mixing.4.Incubate at 56 ℃ for 15 minutes until the sample is completely dissolved. Incubate at 80 ℃ for 15 minutes. Short centrifugation allows the solution on the tube wall to be collected to the bottom of the tube.Note: 1) The purpose of this step is to repair nucleic acids denatured by formaldehyde. Incubating at a high temperature or for too long may cause RNA breakage, resulting in RNA fragments.2) The sample incubated at 56 ℃ can be placed at room temperature until the temperature of the water or dry bath reaches 80 ℃, and then the sample can be incubated at 80 ℃.5. Place on ice for 3 minutes, centrifuge at 12000 rpm for 15 minutes, transfer the supernatant to a new centrifuge tube, be careful not to suck sediment.6. Add 320 to the supernatant µ L Buffer GL, vortex oscillation thoroughly mixed.7. Join 720 µ Mix anhydrous ethanol thoroughly with vortex oscillation.Attention: After adding anhydrous ethanol, there may be a small amount of precipitate precipitation, but it does not affect subsequent operations.8. Add all the solutions obtained in step 7 to the spin columns RS that have been loaded into the collection tube. If the solution cannot be added at once, it can be transferred multiple times. Centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.Optional steps: If genomic DNA needs to be removed, the following steps can be followeda. Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 1 minute, discard the waste liquid, and place the adsorption column back into the recovery manifold.b. Preparation of DNase I mixture: Take 52 µ Add 8 RNase Free Water to it µ 10 x Reaction Buffer and 20 µ DNase I (1U/ µ l) Mix well and prepare to a final volume of 80 µ The reaction solution of L.c. Add 80 µ l of DNase I mixture directly to the adsorption column and incubate at 20-30 ℃ for 15 minutes.d. Add 350 to the adsorption column µ L Buffer RW1, centrifuge at 12000 rpm for 1 minute, discard the waste liquid, and place the adsorption column back into the recovery manifold.9. Add 500 to the adsorption column µ Buffer RW2 (check if anhydrous ethanol has been added before use), centrifuge at 12000 rpm for 1 minute, discard the waste liquid in the collection tube, and place the adsorption column back into the collection tube.10. Repeat step 9.Centrifuge at 11.12000 rpm for 2 minutes and discard the waste liquid from the collection tube. Place the adsorption column at room temperature for a few minutes to thoroughly air dry.Note: The purpose of this step is to remove residual ethanol from the adsorption column, which will affect subsequent enzymatic reactions (such as enzyme digestion, PCR, etc.).12. Place the adsorption column in a new RNase free centrifuge tube, and add 20-50µl to the middle of the adsorption column in the air Place RNase Free Water at room temperature for 2-5 minutes, centrifuge at 12000 rpm for 1 minute, collect RNA solution, and store RNA at -20 ℃.Note: 1) The volume of RNase Free Water should not be less than 20 µ l. Small volume affects the recovery rate. 2) If you want to increase RNA production, you can use 20-50 µ Repeat step 12 for the new RNase Free Water.3) If you want to increase the RNA concentration, you can add the obtained solution back to the adsorption column and repeat step 12... Read More | Product contentComponentY665957-1mlY665957-5ml2×GoldStar Probe Mixture1 ml5×1 mlProbe Primer Mix300 µl5×300 µlHuman DNA Standard（100 ng/µl）100 µl5×100 µl50×High ROX40 µl200 µlProduct IntroductionThis product is a real-time Product contentComponentY665957-1mlY665957-5ml2×GoldStar Probe Mixture1 ml5×1 mlProbe Primer Mix300 µl5×300 µlHuman DNA Standard（100 ng/µl）100 µl5×100 µl50×High ROX40 µl200 µlProduct IntroductionThis product is a real-time fluorescence quantitative PCR kit for detecting the concentration of human male Y chromosome, including carefully optimized PCR reaction solution, primer mixture and standards, especially suitable for the quantitative detection of precious and micro DNA samples. The kit adopts a new efficient and fast hot-start amplification enzyme GoldStar Taq DNA Polymerase, which effectively avoids non-specific amplification caused by non-specific binding of primers and templates or primer dimerization at room temperature. This product realizes accurate quantification of Y chromosome and can be applied in various fields such as genetic mapping, species polymorphism research, disease gene localization, paternity testing and forensic analysis.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used in Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument.Instruments that do not require ROX calibration: Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96, etc.Instruments requiring Low ROX calibration: ABI Prism7500/7500 Fast, QuantStudio®3 System, QuantStudio®5 System, QuantStudio®6 Flex System, QuantStudio®7 Flex System, ViiA 7 System, Stratagene Mx3000/Mx3005P, Corbett Rotor Gene 3000, and others.Instruments requiring High ROX calibration: ABI Prism7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, etc.Note: High Rox and Low Rox are formulated as described in Method of Use 3.Scope of applicationThis product is suitable for quantitative testing of male Y chromosome DNA in scientific research, clinical, forensic medicine and paternity testing.Usage1. Amplification template preparationThe library samples to be detected were diluted with TE (10 mM Tris-Cl, pH 8.0, 1 mM EDTA), and the concentration after dilution was as close as possible to the range of 0.05-10 ng/µL. 4°C on ice was set aside.2. Standard dilution: according to the following table, firstly dilute Human DNA Standard (100ng/uL) with TE to make 5 standards of different concentrations according to the table below. 10ng/µL of DNA Standard 1 (Std.1) can be stored stably at -20℃ for 1 month; Std2-5 can only be used on the same day, and should be placed at 4℃ or on ice when not in use for the time being after preparation. When Std2-5 are not used temporarily after preparation, they should be stored at 4℃ or on ice.Standard sampleCorresponding concentration(ng/µl)Minimum Dilution Volume (Unit：µl）Std.11010 [100 ng/µl DNA Standard]+ 90 TEStd.22.520 [Std. 1] +60 TEStd.30.62520 [Std. 2] +60 TEStd.40.1562520 [Std. 3] +60 TEStd.50.039062520 [Std. 4] +60 TE3. qPCR reaction system preparationBefore preparation, the cryopreserved reagents to be used were completely melted and mixed by inverting several times, then centrifuged briefly and prepared. Standards and templates were diluted as described above and prepared.The base reaction system for 20 µL was as follows:Reagent20 µl Reaction system2×GoldStar Probe Mixture10 µlProbe Primer Mix3 µlTemplate4 µlddH₂O3 µlNote: High ROX model: add 1 µL of 50×High ROX per 50 µL of reaction system; Low ROX model: add 1 µL of 50×High ROX per 500 µL of reaction system.A sufficient amount of reaction system mixture was prepared according to the need, and after the reaction system was prepared and mixed thoroughly, it was added to the reaction wells in a volume of 16 µl per well. Then add the prepared standards and diluted samples into the corresponding reaction wells, the amount of addition is 4µL/well. TE was added to the blank control tube, and the same amount was added at 4 µL/well.It is recommended to use 20 µL for the reaction, if you need to perform a smaller system reaction, reduce the system components in equal proportion.4. qPCR reaction programThe PCR mix of this kit contains a FAM fluorescent probe for the target gene and a VIC fluorescent probe with internal reference to Internal PCR Control (IPC). qPCR program with dual fluorescence of hydrolyzed probes needs to be selected for the assay. Please follow the instructions of the instrument used to set up the qPCR program, and the PCR temperature conditions are as follows:1. Standard curve productionThe standard curve was plotted with reference to the Excel sheet for data processing. The correlation coefficient R2 of the standard curve should be not less than 0.98, and the slope should be located between -3.1 and -3.6 when the Ct value is used as the longitudinal coordinate. If the parameters of the standard curve are unreasonable, it is recommended to repeat the experiment.DNA Standard NameDNA Standard Concentration（ng/µL）DNA Standard 110DNA Standard 22.5DNA Standard 30.625DNA Standard 40.15625DNA Standard 50.03906252. Analysis of results and calculation of concentrationsThe Ct difference between experimental replicate wells for FAM signaling of the target gene should be no more than 0.3, otherwise invalid data need to be deleted or the experiment needs to be repeated, do not use Ct outside the valid Ct range of the standard curve to calculate the concentration of the sample.For specific calculations, please refer to the data processing Excel for this product.If the FAM signal is abnormal, the VIC signal of the internal reference Internal PCR Control (IPC) needs to be analyzed to confirm whether the PCR reaction process is abnormal. If the Ct value of the sample null VIC is significantly larger than that of the standard or blank control wells, it means that the sample inhibits the PCR reaction.matters needing attention1. Before testing, these instructions should be read in detail. It should be operated by personnel with professional experience or qualified by training.2. For use, please mix gently by turning up and down, avoid foaming as much as possible, and use it after centrifugation for a short period of time.3. Avoid repeated freezing and thawing of the product, repeated freezing and thawing may degrade the performance of the product.4. When preparing the reaction solution, please use new or non-contaminated tips and centrifuge tubes to prevent contamination as much as possible... Read More |