Western blot analysis was performed with 50 µg mitochondria as previously described [19 (link)]. The primary antibody anti-PaCRD1 (rabbit, dilution 1:5000, peptide: [H]-SKKEKEVVVEEEEGKKKEL-[OH], Davids Biotechnologie GmbH, Regensburg, Germany) was used. As secondary antibodies, IRDye® 680RD anti-rabbit (goat, 1:15,000 dilution, LI-COR Biosciences, Bad Homburg, Germany, 926-68071) and IRDye® 800CW anti-rabbit (goat, 1:15,000 dilution, LI-COR Biosciences, Bad Homburg, Germany, 926-32211) were used. The Odyssey® Fc imaging system (LI-COR Biosciences, Bad Homburg, Germany) was used for detection. Densitometric quantification was performed with the manufacturer’s software Image Studio Lite (Version 5.2).
Anti rabbit irdye 680rd
Manufactured by LI COR
Sourced in Germany, United States
The Anti-rabbit IRDye 680RD is a near-infrared fluorescent dye that can be used for detecting and quantifying rabbit-derived proteins in various applications such as Western blotting, immunohistochemistry, and flow cytometry. It provides a sensitive and specific signal for the detection of rabbit-derived targets.
Automatically generated - may contain errors
Lab products found in correlation
Anti mouse irdye 800cw, by LI COR (8 mentions)
Anti rabbit irdye 800cw, by LI COR (4 mentions)
Odyssey fc imaging system, by LI COR (3 mentions)
Erk1 2, by Cell Signaling Technology (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
Irdye 800cw, by LI COR (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Image studio lite, by LI COR (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Anti mouse irdye 680rd, by LI COR (2 mentions)
Rabbit anti mek1 2, by Cell Signaling Technology (1 mentions)
Mouse anti mmp9, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti phospho mek1 2 ser217 221, by Cell Signaling Technology (1 mentions)
Mouse anti mmp7, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti actin, by Merck Group (1 mentions)
Rabbit anti gapdh, by Cell Signaling Technology (1 mentions)
P mek1 2, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Gapdh, by Merck Group (1 mentions)
P erk, by Santa Cruz Biotechnology (1 mentions)
17 protocols using anti rabbit irdye 680rd
1
Western Blot Analysis of PaCRD1 Protein
2
Molecular Markers in Neurodegeneration
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We used the following primary antibodies: rabbit anti-mBdnf and proBdnf (Santa Cruz Biotechnology, sc-546), rabbit anti phospho-MEK1/2 (Ser217/221) (Cell Signaling Technology, #9121), rabbit anti MEK1/2 (Cell Signaling Technology, #9122), rabbit anti-Actin (A2066; Sigma-Aldrich), rabbit anti-Hprt (ab10479; Abcam), rabbit anti-Gapdh (Cell Signaling; 2118), HRP anti-rabbit (P0448; Dako), mouse anti-Mmp7 (Santa Cruz; sc-515703), mouse anti-Mmp9 (Santa Cruz; sc-393859), mouse anti-Mmp3 (Santa Cruz; sc-21732), mouse anti-Furin (Santa Cruz; sc-133142), mouse anti-Plasminogen (Santa Cruz; sc-376324), mouse anti-Timp1 (Santa Cruz; sc-365905), rabbit anti-PC1/3 (Cell Signaling; 11914), IRDye 800CW anti-mouse (LI-COR; 925–32210) and IRDye 680RD anti-rabbit (LI-COR; 925–68071)
3
Western Blot Analysis of MAPK Pathway
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BRAF (1:1000, Santa Cruz sc-5284), BRAFV600E (1:1000, NewEast BioSciences 26039), CRAF (1:1000, Santa Cruz C-12), MEK1 (1:1000, Millipore Millipore, 07-641), MEK1/2 (1:1000, Cell Signaling 4694), P-MEK1/2 (1:1000, Cell Signaling 9154), ERK1/2 (1:1000, Cell Signaling 4696), P-ERK (1:200, Santa Cruz sc-7383), P-ERK1/2 (1:500, Cell Signaling 4370), ERK1 (1:200, Santa Cruz sc-94), Actin (1:10000, Invitrogen MA5-15739), GAPDH (1:5000, Sigma, G8795), IRDye800CW anti-mouse (1:800, LICOR 926-32210), and IRDye680RD anti-rabbit (1:800, LICOR 926-68071).
4
Western Blot Analysis of Protein Expression
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Protein extracts were made from muscle (hind limb) and brain from each analysable mouse. Tissues were homogenised in 0.32 M sucrose using a bead homogeniser and centrifuged at 10,000 g at 4 °C for 10 minutes. The supernatant was removed and mixed 1:1 with Laemmli buffer. Samples were boiled at 95 °C for 10 minutes, 10% (v/v) DTT added then loaded onto a 10% polyacrylamide gel and run at 120 V for 1.5 hours. The gel was then transferred at 100 V for 1 hour onto a PDVF membrane. Membranes were incubated in 7% acetic acid and 10% methanol for 15 minutes, washed 4 times for 5 minutes in dH2O and incubated in SYPRO Ruby stain for 15 minutes. Total protein was then imaged on the Odyssey Fc Imaging System (LI-COR) and membranes were blocked for 1 hour in Odyssey blocking buffer (LI-COR). Blots were incubated with primary antibodies for eEF1A2 (Rabbit; Proteintech; 1:500) and GAPDH (Mouse, Millipore, 1:1000) in Odyssey blocking buffer at 4 °C overnight then washed in PBST 3 × 5 minutes and incubated in IRdye 800CW anti-mouse (LI-COR) or IRdye 680RD anti-rabbit (LI-COR) secondary antibodies at 1:5000 for 1 hour at RT. Images were analysed using ImageJ software to assess band intensity and values expressed relative to the loading control. Differences in expression between mice with the G70S/−, − / − and +/+ genotypes were assessed by one way ANOVA with tukey post-hoc testing.
5
Western Blot Protein Analysis Protocol
6
Signaling Pathways in Cancer Cells
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Dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT), propidium iodide (PI), mitotane (1,1-(dichlorodiphenyl)-2,2-dichloroethane (o,p’-DDD)), and EF24 were purchased from Sigma Aldrich, Italy. DMEM-F12, 0.05% trypsin-EDTA, insulin, transferrin, selenium, and antibiotics were from ThermoFisher Scientific, Italy. The primary antibodies used were Akt (cod. 9272), phospho-Akt (Ser473) (cod. 9271), Erk1/2 (cod. 4695), phospho-Erk1/2 (Thr202/Tyr204) (cod. 4370), NF-κB p65 (cod.8242), and phospho-NF-κB p65 (cod.3033), all from Cell Signaling Technology, USA; GAPDH (cod.8245), β-catenin (cod.32572), and phospho-β-catenin (cod. 81305) from Abcam, UK. The secondary antibodies used were IRDye 800 CW anti-mouse and IRDye 680 RD anti-rabbit (LiCor, Lincoln, OR, USA).
7
Western Blot Analysis of NF1 Protein
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Cells were washed twice with chilled PBS and lysed with RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl (Merck), 1mM EDTA (Sigma), 0.5% Igepal CA-630 (Sigma)) supplemented with 3mM DTT (Thermo Fisher), 1mM PMSF (Sigma), 1mM sodium orthovanadate (Sigma), 5mM NaF (Sigma), 10 ug/ml leupeptin (Sigma), 5ug/ml aprotinin (Sigma) and 1xPhosSTOP (Roche). Lysates were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Lysates were boiled with 1X Laemmli buffer (50% glycerol (v/v) (Sigma), 10% SDS (m/v) (Merck) , 0.05% bromophenol blue (m/v) (Sigma), 25% Tris HCl 1M pH 6.8 (v/v) (Millipore), 5% betamercaptoethanol (v/v) (Sigma) in distilled water) and 90 mg of protein was subjected to SDS-PAGE and transferred onto PVDF membranes (18 hours 90 mA at 4 C). Membranes were blocked with Odyssey Blocking Buffer (TBS, LI-COR) and incubated with rabbit anti-NF1 Antibody (Bethyl laboratories) at 4 C overnight; and with mouse anti-atubulin (Sigma) 1 hour at room temperature. Membranes were then incubated with IRDye 680RD anti-Rabbit and IRDye 800CW anti-Mouse secondary antibodies (1:10,000 each, LI-COR) for 1 hour at room temperature and scanned with the Odyssey CLx (LI-COR) using the Image Studio Lite (LI-COR).
8
Western Blot Analysis of Mitochondrial Proteins
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Western blot analysis was performed with 50 µg mitochondria or total protein extract as previously described (Warnsmann et al., 2021) (link). The following primary antibodies were used: anti-GFP (mouse, 1:10000 dilution, Sigma-Aldrich, G6795), anti-PaIAP (rabbit, dilution 1:2500, peptide: As secondary antibodies IRDye ® 680RD anti-rabbit (goat, 1:15000 dilution, LIC-OR Biosciences, Bad Homburg, Germany, 926-68071), IRDye ® 800CW anti-rabbit (goat, 1:15000 dilution, LIC-OR Biosciences, Bad Homburg, Germany, 926-32211) and IRDye® 680RD anti-mouse (goat, dilution 1:15000, LI-COR Biosciences, Bad Homburg, Germany, 926-68070) were used. For detection the Odyssey ® Fc imaging system (LIC-OR Biosciences, Bad Homburg, Germany,) was used and densitometric quantification was performed with the manufacturer's software image studio (Version 5.2).
9
Western Blot Analysis of Deubiquitinases
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Protein lysates were generated from cell pellets with Cell Lysis Buffer (Cell Signaling Technology, Frankfurt, Germany) containing Halt Protease and Phosphatase Inhibitor (Thermo Fisher) by incubating for 10 min on ice and 2 x 5 min in an ultrasonic-water bath. 40 μg of the protein lysate were used for a SDS-PAGE (4–12% gradient gel) via the Bolt Western Blot system and a subsequent western blot performed with the iBlot system (both Thermo Fisher), according to the manufacturers' protocols. Following the blotting, blocking for 3 hours was conducted. The antibodies used afterwards were USP14 (A300-919A, rabbit polyclonal) (1:5000, Bethyl laboratories, Montgomery, Texas, USA), UCHL5 (H110, rabbit polyclonal) (1:400, Santa Cruz Biotechnology, Heidelberg, Germany) and cofilin (clone D3F9) (1:2000, Cell Signaling Technology). For detection in a LI-COR Odyssey imaging reader, anti-rabbit IRDye 680RD (1:20000) and IRDye 800CW (1:20000) secondary antibodies (both LI-COR Bioscience, Bad Homburg, Germany) were employed.
10
Immunoblot Analysis of Protein Extracts
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Protein extracts from log‐phase cultures were prepared using a FastPrep‐24 machine (MP Biomedicals) and separated by 4–12% SDS–PAGE. Immunoblot analysis was carried out using anti‐myc (9E10, Sigma) and anti‐H3 (ab1791, Abcam) primary antibodies. A secondary infrared imaging system (Li‐Cor) was used for detection, with Odyssey goat anti‐mouse IRDYE 800CW and anti‐rabbit IRDYE 680RD (both Li‐Cor) as secondary antibodies.
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