Binding buffer

Manufactured by Promega

Binding buffer is a solution used in various molecular biology techniques to facilitate the binding of biomolecules, such as proteins or nucleic acids, to a solid support or matrix. It provides the necessary conditions for efficient and specific binding, which is a crucial step in many purification and isolation procedures.

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Lab products found in correlation

Trizol, by Thermo Fisher Scientific (1 mentions) Rnaseout, by Promega (1 mentions) Protein g dynabead, by Thermo Fisher Scientific (1 mentions) Pcdna3 vector, by Promega (1 mentions) Lysing matrix e tube, by MP Biomedicals (1 mentions) Maxwell rsc instrument, by Promega (1 mentions) Lysis buffer, by Promega (1 mentions) Microspin g 25 column, by GE Healthcare (1 mentions) T4 polynucleotide kinase, by Promega (1 mentions) Gel shift assay system, by Promega (1 mentions) Bio imaging system, by Syngene (1 mentions) γ 32p atp, by PerkinElmer (1 mentions) Microspin g25, by GE Healthcare (1 mentions)

7 protocols using binding buffer

RNA-Protein Interaction Assay using GST-HuD and His-ZBP1

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Purified GST-HuD and His-ZBP1 proteins were probed with Dynabead Protein G (Life Technologies) conjugated with anti-GST and anti-His antibody (Life technologies). Beads conjugated with mouse IgG were used to see the background binding. Beads were washed with binding buffer (Promega) and incubated with 1 μg of total RNA isolated from rat dorsal root ganglion (DRG) in binding buffer (10 mM HEPES, 3 mM MgCl₂, 40 mM KCl, 1 mM DTT, 400 U of RNaseOUT™ and 5% glycerol) at 4°C for 30 min. After washing, beads were incubated in TRIzol (Life Technologies) and extracted by phenol–chloroform to isolate RNA. RNA was precipitated with ethanol using a glycogen carrier.

Kim H.H., Lee S.J., Gardiner A.S., Perrone-Bizzozero N.I, & Yoo S. (2015). Different motif requirements for the localization zipcode element of β-actin mRNA binding by HuD and ZBP1. Nucleic Acids Research, 43(15), 7432-7446.

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Quantifying Tat-TAR Interaction Dynamics

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Subtype B TAR was cloned between HindIII and BamHI site in pCDNA3 vector (Promega). ³²P-labeled TAR was transcribed in vitro using T7 RNA polymerase. TAR was incubated with increasing amounts of purified Tat protein (0.1–2 μg) for 10 min on ice, followed by 10 min at 37°C with binding buffer (Promega). The reaction was stopped by adding 4X gel loading buffer and Tat variants with TAR complexes were analyzed on 4% Non-denaturing polyacrylamide gels and autoradiography was done. 1 μg of the empty pCMV-myc empty vector was used as a control and the expression of Tat–TAR binding was normalized with interaction with empty vector; the experiment was repeated three times.

Ronsard L., Ganguli N., Singh V.K., Mohankumar K., Rai T., Sridharan S., Pajaniradje S., Kumar B., Rai D., Chaudhuri S., Coumar M.S., Ramachandran V.G, & Banerjea A.C. (2017). Impact of Genetic Variations in HIV-1 Tat on LTR-Mediated Transcription via TAR RNA Interaction. Frontiers in Microbiology, 8, 706.

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Fecal DNA Extraction Using Maxwell RSC

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DNA was extracted using the Maxwell RSC Instrument (Promega) and a prototype version of the Maxwell Fecal Microbiome Kit (Promega), and 250 mg of sample were diluted in 1 mL of lysis buffer (Promega) and heated for 5 min at 95 °C. Samples underwent bead-beating twice at 5.5 m/s for 30 s in Lysing Matrix E tubes (MP Biomedicals, Irvine, CA, USA) and centrifuged at 10,600× g for 5 min. Then, 300 µL of supernatant was added to 300 µL of binding buffer (Promega) and loaded into a Maxwell RSC cartridge containing magnetic beads for DNA purification on the Maxwell RSC Instrument, according to Technical Manual TM473.

Sanchez-Cid C., Guironnet A., Wiest L., Vulliet E, & Vogel T.M. (2021). Gentamicin Adsorption onto Soil Particles Prevents Overall Short-Term Effects on the Soil Microbiome and Resistome. Antibiotics, 10(2), 191.

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Nuclear Extract Transcription Factor Binding Assay

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Double stranded oligonucleotides were annealed and labeled using T4 polynucleotide kinase (Promega, Madison, WI) and [γ-32p] deoxy (d)-ATP (PerkinElmer, Waltham, MA; 3000 Ci/mmol). Labeled oligonucleotides were purified using MicroSpin G-25 columns (GE Healthcare, UK). Five micrograms of nuclear extract were incubated at room temperature in a 20 µl reaction volume for 20 minutes in binding buffer from (Promega). Labeled oligonucleotides, about 35 fmol of [γ-32p] dATP-labeled probe, were added and incubated for another 20 minutes at room temperature. The DNA sequences of the double-stranded oligonucleotides used for this study are listed in Table 1. For competition experiments, a 10-fold molar excess of unlabeled oligonucleotides were added to the binding reaction. Samples were subjected to electrophoresis at a 6% non-denaturing polyacrylamide gel at 250 V for 2–3 hours to separate the DNA-complex from the free oligonucleotides. The gel was pre-run in 5X Tris-borate-EDTA buffer for 30 minutes before loading the samples. Gels were dried by heated vacuum and exposed to film (Eastman Kodak, Rochester, NY) for 4–7 days at −80°C. For supershift assays, polyclonal antibodies against Stat3 (K-15: sc-483) (Santa Cruz Biotechnology) were added to the reaction mixture and incubated for 30 min at room temperature prior to the addition of the radio labeled oligonucleotides.

Rayyan N.A., Zhang J., Burnside A.S, & Good D.J. (2014). Leptin Signaling Regulates Hypothalamic Expression of Nescient helix-loop-helix 2 (Nhlh2) Through Signal Transducer and Activator 3 (Stat3). Molecular and cellular endocrinology, 384(0), 134-142.

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DNA Binding Assays for Transcription Factors

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DNA binding assays were carried out using standard procedures Sambrook et al. [19 ]. Trh and Tgo protein were synthesized in a coupled rabbit reticulocyte lysate transcription-translation system (Promega). For Tgo, we used the cDNA pOT2/LD32037 and T7 RNA polymerase; for Trh, we used pFLC1/RH17284 and T7 RNA polymerase. Unprogrammed lysate (lacking the expression plasmids but containing the RNA polymerase) was used as a negative control for binding assays. Double-stranded DNA probes were synthesized by annealing complementary oligonucleotides that spanned both consensus CME binding sites (see Table 1 for sequences). To radioactively label the probes, the 5’-gg overhangs, created following annealing, were filled in using Klenow enzyme and 32P-dCTP. Binding assays were carried out in binding buffer (Promega Corp.) on ice for 45 minutes, before bound and unbound reaction components were resolved at 4°C on a non-denaturing acrylamide gel, and visualized by autoradiography. For the competition assays, 100-fold excess of non-radioactive dsDNA oligonucleotide was used.

Lovato C.V., Lovato T.L, & Cripps R.M. (2019). Crossveinless is a direct transcriptional target of Trachealess and Tango in Drosophila tracheal precursor cells. PLoS ONE, 14(6), e0217906.

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Gel Shift Assay for Transcription Factor Binding

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A commercial kit, Gel Shift Assay System (Promega), was used. IP proteins used for EMSA were prepared as described above. The protein level of each IP protein was confirmed by Western blot. A reaction mixture for EMSA contained 1 × 10⁵ cpm/μL of radiolabeled double-stranded DNA probe (ccl2/p53oligo), 1 mM DTT, 1 μg each of IP protein (omitted from control), 2 μL of 5× binding buffer (Promega), and nuclease-free water to achieve a final volume of 10 μL. Mixtures were added with cold competitor or T7 primer as control and incubated at room temperature for 20 min, followed by electrophoresis on nondenaturing 6 % polyacrylamide gels in Tris-borate-EDTA buffer [90 mmol/L Tris-borate/2 mmol/L EDTA HEPES (pH 8)]. The gel was dried and exposed to a SynGene bio imaging system (Frederick, MD). The signal was further improved by Photoshop (Adobe).

Tang X, & Amar S. (2014). p53 suppresses CCL2-induced subcutaneous tumor xenograft. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, 36(4), 2801-2808.

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Electrophoretic Mobility Shift Assay of NRF-1 Binding

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Oligonucleotide probes containing the NRF-1 binding site on the GluR2 promoter (Table S1) were annealed, and with [γ³²P] ATP (3000 Ci/mmol at 10 mCi/mL; Perkin-Elmer, Shelton, CT, USA). The oligonucleotides were then separated from the unincorporated nucleotides by chromatography using a Micro Spin G-25 column (GE Healthcare, Waukesha, WI, USA). The purified probes were incubated with 5 μg of nuclear extract, 5× binding buffer (Promega), and gel loading buffer. For competition, oligonucleotides were incubated with the nuclear extract before adding the oligonucleotides. The probe/nuclear extract mixture was loaded onto a 4% polyacrylamide gel, and run at 200 V for 60–90 min. The results were visualized by autoradiography.

Ishida K., Aoki K., Takishita T., Miyara M., Sakamoto S., Sanoh S., Kimura T., Kanda Y., Ohta S, & Kotake Y. (2017). Low-Concentration Tributyltin Decreases GluR2 Expression via Nuclear Respiratory Factor-1 Inhibition. International Journal of Molecular Sciences, 18(8), 1754.

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