Ahp500g

Manufactured by Bio-Rad

Sourced in United Kingdom

The AHP500G is a power supply unit designed for electrophoresis applications. It provides a constant voltage or current output to power various electrophoresis equipment. The device features adjustable voltage and current settings, as well as display of the current operating parameters.

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Lab products found in correlation

5 protocols using ahp500g

Antibody Panel for Endosomal Trafficking

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Antibodies used in this study are as follows: SNX1 [clone 51; 611482; BD Bioscience; immunofluorescence (IF) 1:200], GLUT1 (ab40084; Abcam; IF 1:200), Golgin97 (clone CDF4; A-21270; Thermo Fisher Scientific; IF 1:400), VPS26 (ab23892; Abcam; IF 1:200), VPS35 (ab10099; Abcam; IF 1:200), VPS35 [ab97545; Abcam; IF 1:200), VPS35 [ab157220; Abcam; western blotting (WB) 1:1000], VPS29 (ab98929; Abcam; WB 1:200), FAM21 (gift from Daniel D. Billadeau, Mayo Clinic, Rochester, MN; IF 1:400), EEA1 (N-19; sc-6415; Santa Cruz Biotechnology; IF 1:200), TGN46 (AHP500G; Bio-Rad Laboratories; IF 1:200), anti-Myc (gift from Harry Mellor, The University of Bristol, UK; IF 1:500), LAMP1 (DSHB Hybridoma Product; H4A3; deposited to the DSHB by August, J.T./Hildreth, J.E.K.; IF 1:500), LAMP1 (ab24170; Abcam; IF 1:200), mouse EEA1 (610457; BD Bioscience; IF 1:200), CI-MPR (ab124767; Abcam; WB 1:1000, IF 1:200), Itgβ1 (TS2/16; IF 1:200), SNX6 (Clone D-5; 365965; Santa Cruz Biotechnology; WB 1:500), PMP70 (PA1-650; Invitrogen; IF 1:200), WASH1 (gift from Daniel D. Billadeau; IF 1:400), C16orf62 (PA5-28553; Pierce; IF 1:200), sortilin (ab16640; Abcam; WB 1:1000), cathepsin D (21327-1-AP, Proteintech; WB 1:1000), SNX5 (ab180520; Abcam; WB 1:500) and β-actin (A1978; Sigma-Aldrich; WB 1:2000).

Evans A.J., Daly J.L., Anuar A.N., Simonetti B, & Cullen P.J. (2020). Acute inactivation of retromer and ESCPE-1 leads to time-resolved defects in endosomal cargo sorting. Journal of Cell Science, 133(15), jcs246033.

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Immunofluorescence Analysis of AP4 Complex

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The following reagents were used: Bovine serum albumin (AmericanBIO, #9048–46-8), saponin (Sigma, #47036-50G-F), Molecular Probes Hoechst 33258 (#H3569) and Alexa Fluor 647-labelled phalloidin (Thermo Fisher Scientific, #A22287). The following primary antibodies were used: Anti-ATG9A at 1:400 (Abcam # ab108338), anti-TGN46 at 1:800 (Bio-Rad #AHP500G), anti-AP4E1 and anti-AP4B1 (both produced in house⁶ (link)). Fluorescently labelled secondary antibodies used in this study were all purchased from Thermo Fisher Scientific and used at 1:2000 (#A11008, #A11016, #A21245). Horseradish peroxidase-conjugated secondary antibodies were purchased from Sigma-Aldrich and used at 1:5000 dilution. Lentivirus to express human AP4B1 under a PGK promoter was generated as described previously.¹¹ (link)

Ebrahimi-Fakhari D., Alecu J.E., Brechmann B., Ziegler M., Eberhardt K., Jumo H., D’Amore A., Habibzadeh P., Faghihi M.A., De Bleecker J.L., Vuillaumier-Barrot S., Auvin S., Santorelli F.M., Neuser S., Popp B., Yang E., Barrett L., Davies A.K., Saffari A., Hirst J, & Sahin M. (2021). High-throughput imaging of ATG9A distribution as a diagnostic functional assay for adaptor protein complex 4-associated hereditary spastic paraplegia. Brain Communications, 3(4), fcab221.

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Immunofluorescence Staining of Golgi and Membrane Proteins

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Cells were grown on glass coverslips for one day, then fixed with 3% PFA for 10 min, quenched with 50 mM NH4Cl in PBS for 5 min, permeabilized with 0.2% TritonX-100 in PBS for 10 min, blocked with 1% BSA in PBS for 1 h, and incubated with primary antibodies diluted in 1% BSA in PBS for 1 h: Anti-AP1g1 (1:1000, self-made from hybridoma cells), anti-bCOP (1:500, CM1, hybridoma supernatant, gift from Dr. Felix Wieland, Heidelberg University), anti-GGA2 (1:500, BD Bioscience 612613), anti-GM130 (1:1000, Cell Signaling 12480S), and anti-TGN46 (1:1000, BioRad AHP500G). Samples were washed and incubated with fluorescent secondary antibodies diluted in 1% BSA in PBS for 1 h (1:400; anti-mouse-Alexa488, Invitrogen A21202; anti-rabbit-Alexa568, Invitrogen A10042; anti-sheep-Cy3, Jackson ImmunoResearch 713-165-147). Coverslips were mounted in FluoromountG (SouthernBiotech) supplemented with 0.5 ng/ml DAPI (Sigma-Aldrich) and stored in the dark at 4°C. For localization of GFP-KDEL, cells were grown on coverslips for one day, transfected with the pcDNA3-ss-GFP-KDEL, and fixed and stained a day later.

Pennauer M., Buczak K., Prescianotto‐Baschong C., & Spiess M. (2021). Shared and specific functions of Arfs 1–5 at the Golgi revealed by systematic knockouts.

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COMP Protein Immunofluorescence and STORM Analysis

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The commercial antibodies used in this study, and their sources, are as follows: Anti-COMP (rabbit polyclonal, ab74524; Abcam), anti-FLAG (mouse monoclonal, F3165; Sigma-Aldrich), and anti-TGN46 (sheep polyclonal, AHP500G; AbD Serotec) were used for WB, together with the secondary antibodies sheep (sc-2770, sc-2303, rabbit sc-2301 and mouse sc-2314) obtained from Santa Cruz Biotechnology, Inc. Anti-HA (rat monoclonal, 11867423001; Roche), anti-FLAG (mouse monoclonal; F3165; Sigma-Aldrich), anti-GM130 (mouse monoclonal, 610822; BD Biosciences), and anti-ATP2C1 (mouse monoclonal, HPA035116; Sigma-Aldrich) were used for immunofluorescence microscopy. The secondary antibodies Alexa Fluor 488 rabbit (A21206), Alexa Fluor 594 mouse (A21203), and Alexa Fluor 488 rat (A21208) were purchased from Thermo Fisher Scientific. For STORM measurements Cy5-conjugated AffiniPure (rabbit, 711–175-152; Jackson ImmunoResearch Laboratories, Inc.) was used for Cab45 as a secondary antibody and Alexa Fluor 488 sheep (A11015) for TGN46 (Thermo Fisher Scientific). The recombinant proteins COMP-FLAG (COMP-3659H; Creative BioMart), COMP (ab104358; Abcam), LysozymeC (Sigma-Aldrich), and LysozymeC-Cy5 (LS1-S5-1; Nanocs) were used for in vitro analysis.

Crevenna A.H., Blank B., Maiser A., Emin D., Prescher J., Beck G., Kienzle C., Bartnik K., Habermann B., Pakdel M., Leonhardt H., Lamb D.C, & von Blume J. (2016). Secretory cargo sorting by Ca2+-dependent Cab45 oligomerization at the trans-Golgi network. The Journal of Cell Biology, 213(3), 305-314.

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Immunofluorescence Analysis of APP and Organelle Markers

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Hela cells grown on coverslips were fixed in paraformaldehyde 4% solution for 10 min, permeabilized with Triton-X 100 (0.1%) for 10 min, saturated in BSA (5%)/Tween20 (0.1%), and probed for 1 h with appropriate primary antibodies: APPCter rabbit polyclonal (1:5000), APPCter, mouse monoclonal (1:5000, Biolegend), WO2 mouse monoclonal (1:5000), or ηCTF-Nter rabbit polyclonal (1:1000) for ηCTF detection, α-Lamp2 mouse monoclonal (1:1000, Santa Cruz), α-EEA1 rabbit monoclonal (1:200, Cell Signaling Technology), α-CD63 mouse monoclonal (1:1000, Santa Cruz) and α-TGN46 sheep polyclonal (Serotec, AHP500G, 1:1000) for lysosomes, early and late endosomes and golgi specific markers, respectively. After washes, coverslips were incubated for 1 h with Alexa Fluor-488 and Alexa Fluor-590 conjugated antibodies (Molecular Probes, 1:1000) and DAPI (1:20,000, Roche) staining. Finally, the sections were washed with PBS, then mounted onto glass slides and cover-slipped. The stained slices were kept at 4◦C before analysis with confocal microscopy (Zeiss LSM 780 with 63 × Objective).

Afram E., Lauritzen I., Bourgeois A., El Manaa W., Duplan E., Chami M., Valverde A., Charlotte B., Pardossi-Piquard R, & Checler F. (2023). The η-secretase-derived APP fragment ηCTF is localized in Golgi, endosomes and extracellular vesicles and contributes to Aβ production. Cellular and Molecular Life Sciences, 80(4), 97.

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