Hybond lfp membrane

Manufactured by GE Healthcare

Sourced in United States

Hybond LFP membranes are a type of laboratory equipment used in various analytical and experimental procedures. They are designed to facilitate the transfer and immobilization of biomolecules, such as proteins, nucleic acids, and other macromolecules, during blotting techniques. The membranes provide a stable and efficient platform for these applications.

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Lab products found in correlation

Ecl prime detection system, by GE Healthcare (2 mentions) Ecl plus western blotting system, by GE Healthcare (1 mentions) Typhoon 9410, by GE Healthcare (1 mentions) Imagequant software, by GE Healthcare (1 mentions) Ni nta magnetic agarose beads, by Qiagen (1 mentions) Typhoon phosphoimager, by Cytiva (1 mentions) Imagequant software, by Cytiva (1 mentions) Endobulin, by Baxter (1 mentions) Hrp conjugated goat anti human igg, by Merck Group (1 mentions) Hrp conjugated goat anti rabbit igg, by Merck Group (1 mentions) Skimmed milk, by Merck Group (1 mentions) Ac 74, by Merck Group (1 mentions) Typhoon 9410 imager, by GE Healthcare (1 mentions) Imagequant tl software, by GE Healthcare (1 mentions) Typhoon trio, by GE Healthcare (1 mentions) Complete protease inhibitor cocktail, by Roche (1 mentions)

6 protocols using hybond lfp membrane

Western Blot Analysis of SmpB Proteins

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Lysates of 0.02-0.03 A₆₀₀ units of cells were fractionated on a 10% SDS/Tricine-polyacrylamide gel. Proteins were blotted to a Hybond-LFP membrane (GE Healthcare) by a wet transfer in Towbin buffer containing 20% methanol and 0.05% SDS. Rabbit polyclonal antibodies raised against the E.coli SmpB protein and Cy5-labeled anti-rabbit secondary antibodies from ECL-Plex system (Amersham, GE Healthcare) were used to detect both E.coli and M. tuberculosis SmpB proteins. Blots were visualized using Typhoon 9410 or LAS 4100 imagers and quantified using ImageQuant software (GE Healthcare).
SmpB proteins with the Tag-100 at their C-termini were detected with rabbit anti-Tag-100 monoclonal antibody from GenScript and anti-rabbit IgG-HRP using the ECL-Plus Western Blotting System (Amersham, GE Healthcare).

Wower I.K., Zwieb C, & Wower J. (2014). Requirements for resuming translation in chimeric transfer-messenger RNAs of Escherichia coli and Mycobacterium tuberculosis. BMC Molecular Biology, 15, 19.

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In Vitro Tagging of Ribosomal Protein

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All assays were performed with the E. coli T7 transcription/translation system for circular DNA from Promega [22 (link)]. A typical 25-μL reaction mixture contained 2 μg of circular plasmid pETrpmA-At-1 encoding the gene for truncated ribosomal protein L27, 25 pmoles of His-tagged SmpB protein, 40 pmoles of tmRNA, and 20 units of SUPERase-In^TM. Tagging reaction was carried out for 1 h at 37°C. Tagged proteins were captured on Ni-NTA magnetic agarose beads (Qiagen), fractionated on a 10% SDS/Tricine-polyacrylamide gel and blotted to a Hybond-LFP membrane (GE Healthcare Life Sciences). Because His-tagged truncated protein L27 had T7-tag peptide on its N-terminus, it could be detected using the anti-T7 tag monoclonal antibody from Invitrogen and the secondary Cy5-labeled antibody from Amersham. Band intensity was quantified using a Typhoon Phosphoimager and ImageQuant software (Amersham Biosciences).

Wower I.K., Zwieb C, & Wower J. (2014). Requirements for resuming translation in chimeric transfer-messenger RNAs of Escherichia coli and Mycobacterium tuberculosis. BMC Molecular Biology, 15, 19.

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Isolation and Immunoblotting of Streptococcal Cell Wall

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Streptococcal cell wall extracts were isolated as previously described26 (link). Briefly, overnight S. pyogenes cultures were diluted 1:10 in fresh THB and grown to an A₆₀₀ of 0.4–0.8. The cells were pelleted and resuspended in 1/50th volume of 10 mM Tris-HCl (pH 8.0) containing 30% raffinose, 100 U/ml mutanolysin, 1 mg/ml lysozyme and 10% Protease Inhibitor Cocktail Set III (Calbiochem), and incubated for 3 h at 37 °C with occasional inversion. The cells were pelleted and the supernatant (cell wall extract) was dialysed into PBS overnight and concentrated by centrifugation (10,000 MWCO). For immunoblotting, proteins were separated on 7% tris-acetate (TA) gels (Invitrogen) and transferred to Hybond LFP membranes (GE Healthcare). Membranes were blocked with 5% skimmed milk prior to the addition of 4 μg/ml IVIG (Endobulin^®, Baxter). Bound antibodies were detected using a 1:80,000 dilution of HRP-conjugated goat anti human IgG (Sigma-Aldrich) and the ECL prime detection system (GE Healthcare).

Reglinski M., Gierula M., Lynskey N.N., Edwards R.J, & Sriskandan S. (2015). Identification of the Streptococcus pyogenes surface antigens recognised by pooled human immunoglobulin. Scientific Reports, 5, 15825.

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Western Blot Analysis of CTAb Antibody

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Samples were separated on 10% bis-tris gels (Novex) and transferred to Hybond LFP membranes (GE Healthcare). Membranes were blocked with 5% skimmed milk (Sigma-Aldrich) prior to the addition of CTAb antibody^{8 (link)}. Bound antibodies were detected using a 1:80,000 dilution of HRP-conjugated goat anti-rabbit IgG (Sigma-Aldrich) and the ECL prime detection system (GE Healthcare). Where appropriate, TCA-precipitated bacterial supernatants, or recombinant proteins, rSIC^{24 (link)} and rSpeB (Toxin Technology), were used as positive controls.

Edwards R.J., Pyzio M., Gierula M., Turner C.E., Abdul-Salam V.B, & Sriskandan S. (2018). Proteomic analysis at the sites of clinical infection with invasive Streptococcus pyogenes. Scientific Reports, 8, 5950.

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Optimized Western Blot Quantification

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For Western blot analysis, we used rabbit polyclonal antibodies against CypA (sc-20360-R) or CypD (sc-66848) and a goat polyclonal antibody against CypB (sc-20361) (all from Santa Cruz Biotechnology), a rabbit monoclonal antibody against CypC (ab184552, Abcam), and mouse monoclonal antibodies against β-actin (AC-74, Sigma) or transferrin receptor (TfR; H68.4, Thermo Fisher Scientific). Cells were lysed in Laemmli sample buffer and after SDS-PAGE, proteins were transferred to Hybond-LFP membranes (GE Healthcare) by semi-dry blotting. Membranes were blocked with 1% casein in PBS containing 0.1% Tween-20 (PBST), and were incubated in PBST with 0.5% casein with anti-CypA (1:500), anti-CypB (1:1000), anti-CypC (1:1000), anti-CypD (1:500), anti-TfR (1:4000), or anti-β-actin (1:50,000) antisera. Biotin-conjugated swine-anti-rabbit IgG (1:2000) or goat-anti-mouse IgG (1:1000) antibodies (DAKO) and Cy3-conjugated mouse-anti-biotin (1:2500) were used for detection. Blots were scanned with a Typhoon 9410 imager (GE Healthcare) and analyzed with ImageQuant TL software.

de Wilde A.H., Zevenhoven-Dobbe J.C., Beugeling C., Chatterji U., de Jong D., Gallay P., Szuhai K., Posthuma C.C, & Snijder E.J. (2017). Coronaviruses and arteriviruses display striking differences in their cyclophilin A-dependence during replication in cell culture. Virology, 517, 148-156.

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Quantification of Phosphorylated p38 in MCI-treated Cells

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Cells were lysed with 20 mM HEPES-NaOH (pH7.4) containing 0.1% Triton X-100 and 1× Complete Protease Inhibitor Cocktail (Roche, Basel, Switzerland). The proteins were separated by SDS-PAGE and transferred to Hybond-LFP membranes (GE Healthcare, Chicago, IL, USA). The membranes were blocked at room temperature for 1 h in blocking reagent (GE Healthcare) and probed with the primary antibodies. Immunoreactivity was determined using ECL-Plex (GE Healthcare), and images were acquired using Typhoon Trio (GE Healthcare). The intensities of the immunoreactivities were quantified using ImageQuantTL software (GE Healthcare). The intensity of immunoreactivity in each MCI-treated sample was normalized to that in an untreated sample on the same blotting membrane. After the normalized values of total and phosphorylated p38 had been acquired, the ratio of the phosphorylated form to total p38 was calculated in each experiment.

Sakurai H., Yokoyama M., Katsumata-Kato O, & Fujita-Yoshigaki J. (2019). Suppression of parotid acinar cell dysfunction by the free radical scavenger 3-methyl-1-phenyl-2-pyrazolin-5-one. Journal of oral science, 61(3).

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