For western blotting, 10 μg (human central nervous system lysates) or 20 μg (zebrafish lysates) of protein was loaded on a Bis-Tris 4–12% gradient SDS-PAGE (WG1402BOX, Invitrogen, Thermo Fisher Scientific) in MOPS-SDS running buffer (J62847.K2, Alfa Aesar, Haverhill, MA, USA), electrophoresed at 150 V for 60 min, and transferred to a nitrocellulose membrane (GE10600001, Semidry transfer, Biorad, Hercules, CA, USA). Membranes were blocked with 5% non-fat dried milk (A0830.1000, AppliChem, Darmstadt, Germany) in phosphate-buffered saline (PBS) 0.1% Tween-20 (PBST). Primary antibodies and the corresponding dilutions are listed in Supplementary Table 2 (Online resource). Secondary antibodies were goat anti-rabbit IgG-HRP or goat anti-mouse IgG-HRP (1:10 000, P044801-2 and P044701-2, polyclonal, Dako). Blots were developed with SuperSignal West Pico or Dura plus ECL reagent (34580 and 34075, Thermo Fisher Scientific). Digital images were acquired using the Amersham Imager 600 (GE Healthcare, Chicago, IL, USA). All blots were stripped (21063, Restore Western Blot Stripping Buffer, Thermo Fisher Scientific) of bound antibodies and reprobed with GAPDH to control for equal protein loading. Band intensities were measured using ImageJ and were normalized to GAPDH.
P044701 2
Manufactured by Agilent Technologies
Sourced in United States
The P044701-2 is a laboratory equipment product from Agilent Technologies. It is a device designed for use in scientific research and analysis applications. The core function of this product is to perform specific tasks within the laboratory environment, though detailed specifications are not available without further information.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse igg hrp, by Agilent Technologies (1 mentions)
Goat anti rabbit igg hrp, by Agilent Technologies (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Wg1402box, by Thermo Fisher Scientific (1 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
A3854, by Merck Group (1 mentions)
Goat anti mouse horseradish peroxidase hrp conjugated secondary antibody, by Agilent Technologies (1 mentions)
Sc 17792, by Santa Cruz Biotechnology (1 mentions)
Ab2828, by Abcam (1 mentions)
Benzonase, by Thermo Fisher Scientific (1 mentions)
Anti mecp2, by Abcam (1 mentions)
Anti asyn, by BD (1 mentions)
Ecl western blotting detection system, by GE Healthcare (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Chemostar, by Intas (1 mentions)
Chemocam system, by Intas (1 mentions)
Ab6046, by Abcam (1 mentions)
7 protocols using p044701 2
1
Western Blot Analysis of CNS and Zebrafish Lysates
2
Western Blot Analysis of TGFβR2 Protein
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Protein extracts were prepare as described (Link et al., 2006 (link)). TgfβR2 protein was detected by a primary anti-tgfβR2 antibody (diluted 1:250 in blocking solution, sc-17792; Santa Cruz Biotechnology) followed by a goat anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibody (1:1,000 in blocking solution, P044701-2, DAKO). An anti β-actin-HRP-conjugated antibody (1:35,000, A3854; Sigma) was used for loading control.
3
Immunoblotting for α-synuclein and MeCP2
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Protein extracts were prepared by resuspending cells in lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% Triton × 100, 10 mM MgCl 2 , 1× Halt protease inhibitor-cocktail (Thermo Fisher), 1 μl/ml Benzonase (Thermo Fisher). After incubation on ice for 1 h, 50 μg of protein per lane was separated by SDS gel electrophoresis, transferred to nitrocellulose, and detection was performed with the ECL Western blotting detection system (GE Healthcare) and secondary antibodies conjugated with horseradish peroxidase. The following antibodies and dilutions were used: anti-a-syn, 1:2000 (610786, BD Bioscience); anti-MeCP2, 1:1000 (ab2828, Abcam); anti beta-actin, 1:10000 (A5441, Sigma); goat anti-mouse, 1:2000 (P044701-2, DAKO); and goat anti-rabbit, 1:3000 (7074, Cell Signaling). Immunoreactive signals were detected using the Intas ChemoCam system (Intas) and the software ChemoStar (Intas). Quantification of the signal intensities was performed using ImageJ.
4
Fibroblast Protein Extraction and Western Blotting
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Protein extraction: Skin fibroblast cells were collected as mentioned above. Cells were lysed with 1× lysis buffer [50 m Tris-HCl pH7.5; 250 m NaCl; 1 m EDTA; 50 m NaF; 1× Complete Protease Inhibitor, EDTA-free (Roche, REF 11873580001); 1% Triton-X-100 and 0.1 m Na 3 VO 4 ], and proteins were extracted by two rounds of low-frequency sonication for 5 s, followed by quick centrifugation to remove cell debris.
Immunoblotting: Western blot was performed as previously described (36) . Polyclonal anti-AKR1C3 (Abcam; ab27491) used as primary and polyclonal Rabbit anti-goat conjugated to HRP (Dako; P044901-2) as secondary antibody. The blots were reprobed with rabbit polyclonal β-tubulin antibody (Abcam, ab6046) as primary and polyclonal goat anti-Rabbit conjugated to HRP (Dako; P044701-2) as secondary antibody.
Immunoblotting: Western blot was performed as previously described (36) . Polyclonal anti-AKR1C3 (Abcam; ab27491) used as primary and polyclonal Rabbit anti-goat conjugated to HRP (Dako; P044901-2) as secondary antibody. The blots were reprobed with rabbit polyclonal β-tubulin antibody (Abcam, ab6046) as primary and polyclonal goat anti-Rabbit conjugated to HRP (Dako; P044701-2) as secondary antibody.
5
Quantifying Active Rac1 in Intestinal Tissues
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RAC1 activity was determined on snap frozen small intestinal tissue and performed using the Cytoskeleton Rac1-activation assay kit (Cytoskeleton; #BK035) according to the manufacturer’s instructions. Briefly, tissues were lysed with lysis buffer supplemented with protease inhibitors. Around 800 μg of lysates was precleared and PAK-PBD beads were added to pull-down active Rac1. Positive (GTPγS) and negative (GDP) bound control samples were prepared according to standard protocol (Cytoskeleton). Following immunoprecipitation at 4 °C for 1 h, beads were washed 3x in washing buffer, and precipitated proteins detected by polyacrylamide gel electrophoresis and subsequent western blotting. Protein bound membranes were incubated overnight at 4 °C together with primary anti-Rac1 monoclonal antibody (Cytoskeletion; #ARC03; 1:500) in block solution (TBS containing 5% BSA, and 0.02% Triton X-100). After washing the membranes thoroughly in TBS-T, they were incubated 1 h at room temperature with secondary goat mouse antibody (Agilent #P044701-2, 1:2000) in blocking solution. For assessment of total RAC1 in whole cell lysates (20 μg protein/sample), mouse anti-RAC1 antibody (Cytoskeletion; #ARC03; 1:500) was used.
6
Quantifying SERCA1 Protein Levels in Muscle Samples
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Western blot analysis of SERCA1 protein was previously performed in muscle samples from five literature cases. Additionally, we assessed SERCA1 protein content in muscle homogenates from four new patients and one literature patient. Muscle homogenates (2% w/v) were prepared by homogenization using a Potter/Elvehjem tissue homogenizer in 250 mM sucrose, 2 mM EDTA, and 10 mM Tris-HCl (pH 7.6). Protein concentration was determined using the Lowry method. Approximately 20 µg of total protein was loaded per lane on a 10% polyacrylamide gel. Electrophoresis and western blotting were performed following standard laboratory methods, after which SERCA1 protein was detected using anti-SERCA1 monoclonal antibody (ab2819, Abcam, ITK Diagnostics) and a goat-anti-mouse peroxidase-conjugated secondary antibody (P044701-2; Agilent-Dako), followed by chemiluminescent detection. To check for equal loading, blots were stained for GAPDH using anti-GAPDH monoclonal antibody (ab8245, Abcam, ITK Diagnostics).
7
Western Blot Analysis of Cell Signaling
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Cell pellets were lysed in RIPA-buffer supplemented with protease and phosphatase inhibitor. The protein concentration was determined with a Pierce BCA Protein Assay Kit (23227, Thermo Fisher Scientific, Waltham, Massachusetts, USA). Equal amounts of protein were used for gel electrophoresis and blotted on a nitrocellulose membrane. Membranes were incubated at 4 °C overnight with anti-B-ACTIN (1:5 000, A5441, Sigma Aldrich), anti-GSK-3 α/ß (1:200, sc-7291, Santa Cruz Biotechnology, Dallas, Texas, USA), anti-MCL-1 (1:4 000, ab32087, abcam), anit-pGSK-3α/ß Ser21/9 (1:1 000, 8566, Cell Signaling Technology), anti-RPS6KA1 (ab32114, Abcam, Cambridge, UK; 1:1 000) and secondary antibodies against mouse or rabbit immunoglobulin (1:4 000, P044701-2, P044801-2, Dako/Agilent, Santa Clara, California, USA). Proteins were visualized on an Amersham imager 600 (Cytiva, Chalfont St Giles, UK) using ECL reagent. All images have been cropped for improved clarity and conciseness. Densitometry analysis was performed using ImageJ.
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