Immobilised GST-NHSL1-6, 7, 8 (mAb) or GST-aa387-546NHSL1 (pAb) (Fig. 1f ) were digested with Prescission protease (Amersham Pharmacia Biotech) and used to produce monoclonal antibodies by PEG induced fusion of primary B cells isolated from popliteal lymph nodes with the mouse myeloma cell line P3-X63-Ag-8. The NHSL1 monoclonal antibody was subcloned twice (clone C286F5E1; IgG1) and used for western blot: undiluted hybridoma supernatant, immunofluorescence: 50 μg/ml. GST-aa387-546NHSL1 (Fig. 1f ) after digestion with Prescission protease (Amersham Pharmacia Biotech) was used to raise polyclonal rabbit antiserum #4457 (Eurogentec) and used for western blot: 1:2000, immunofluorescence: 1:400. Commercial primary antibodies: EGFP, western blot: 1:2000 (11814460001, Roche); Myc, western blot: 1:5000 (M5546, 9E10, Sigma); MBP, western blot: 1:10,000 (E8032S, New England Biolabs); Abi1, western blot: 1:1000, immunofluorescence: 1:100 (MBL, clone 1B9, D147-3), Scar/WAVE1, immunofluorescence: 1:50 (BD 612276), Scar/WAVE2 rabbit mAb, western blot: 1:1000, immunofluorescence: 1:50 (D2C8, CST 3659), ARPC2, western blot: 1:1000, immunofluorescence: 1:100 (07-227-I-100UG, Millipore). Secondary antibodies: HRP-goat anti-rabbit, western blot: 1:2000 (P044801, Agilent-Dako); HRP-goat anti-mouse, western blot: 1:2000 (P044701, Agilent-Dako).
M5546
Manufactured by Merck Group
Sourced in Japan, United States, United Kingdom
The M5546 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of the M5546 is to provide a controlled environment for various scientific experiments and analyses. Further details on the intended use or specific applications of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
F1804, by Merck Group (2 mentions)
Ab20272, by Abcam (2 mentions)
Prescission protease, by Cytiva (1 mentions)
A11122, by Thermo Fisher Scientific (1 mentions)
G 21040, by Thermo Fisher Scientific (1 mentions)
Flag m2, by Merck Group (1 mentions)
α tubulin dm1a, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Ab48394, by Proteintech (1 mentions)
C myc 9e10, by Merck Group (1 mentions)
R960 25, by Thermo Fisher Scientific (1 mentions)
Ab104232, by Abcam (1 mentions)
P7962, by Merck Group (1 mentions)
Y 180, by Santa Cruz Biotechnology (1 mentions)
A9521, by Merck Group (1 mentions)
Protein a sepharose beads, by GE Healthcare (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ab290, by Abcam (1 mentions)
Ab1424, by Abcam (1 mentions)
10 protocols using m5546
1
Monoclonal Antibody Production and Characterization
2
Maintenance and Antibody Characterization of HEK293T Cells
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Human embryonic kidney (HEK) 293T cells were maintained in complete Dulbecco’s modified Eagle’s medium containing 10% heat-inactivated fetal bovine serum (Sigma Aldrich), 1 mM MEM non-essential amino acids solution, and antibiotics (mixture of 100 U/mL penicillin G sodium and 100 μg/mL streptomycin sulfate). Cells were grown at 37°C in a humidified atmosphere containing 5% CO2. The following antibodies were purchased from commercial sources: c-myc (9E10; Sigma Aldrich M5546), FLAG (M2; Sigma Aldrich F1804, F7425), α-tubulin (DM1A; Sigma Aldrich T6199), p62 (Abnova PAB16850), p62 phospho-Ser 351 (kindly provided by Dr. Komatsu, Tokyo Metropolitan Institute of Medical Science, Japan), p62 phospho-Ser 403 (4F6; Millipore MABC186), LRRK2 [MJFF2(c41-2); Epitomics 3514–1], LC3 (Abcam ab48394), Keap1 (Proteintech 10503-2-AP), green fluorescent protein (GFP, Invitrogen A11122), and horseradish peroxidase (HRP)-conjugated secondary antibodies (Invitrogen G21040).
3
Protein Extraction and Western Blotting
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Lysates for Western blotting were prepared using TCA extraction, as previously described [51 (link)]. Following SDS-PAGE and transfer to nitrocellulose, blots were probed with antibodies against PSTAIRE (P7962, Sigma), V5 (R96025, Invitrogen), MYC (clone 9E10, M5546, Sigma), Clb2 (y-180, Santa Cruz Biotechnology), Rad53 (ab104232, Abcam) or G6PDH (A9521, Sigma) as indicated.
4
Co-Immunoprecipitation in N. benthamiana
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N. benthamiana leaves were collected and ground in liquid nitrogen into powders, and co-IP experiments were performed at 4°C. 1 ml radioimmunoprecipitation assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% Na-deoxycholate, 1% NP-40, 1 mM PMSF, and 1% protease inhibitor cocktail [Sigma-Aldrich]) was used to extract 500 µl of plant tissue. One tenth of the protein extracts was used as the input sample, and the rest were used for IP using protein A–Sepharose beads (GE Healthcare) precoated with a rabbit anti-GFP antibody (ab290; Abcam). After 3× wash using radioimmunoprecipitation assay buffer, the immunoprecipitates and the input samples were separated by 8 or 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories), and detected with a mouse anti-GFP (1:2,000; 632569; Takara Bio Inc.) or a mouse anti-Myc (1:,000; M5546; Sigma-Aldrich) antibody. The input/IP ratio is 1:9.
5
Co-immunoprecipitation Analysis of Trypanosome Proteins
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For co-immunoprecipitation (co-IP) analysis, TbISWI, NLP, RCCP, and FYRP were tagged at the C terminus with either a triple Myc epitope using the pMoTAG43M vector (59 (link)) or with a triple HA epitope using the pMoTAG4H vector (54 (link), 55 (link), 59 (link)) in PF and BF cell lines. Cell extracts were prepared as for the TAP tagging protocol (63 (link)), except that 0.1% Nonidet P-40 was added while extracting protein. Sepharose CL-4B columns (GE Healthcare) were prepared with ice-cold IP buffer (150 mm sucrose, 20 mm l -glutamic acid, 20 mm HEPES-KOH (pH 7.7), 3 mm MgCl2, 1 mm DTT, 150 mm KCl, 0.1% Nonidet P-40) and incubated with either monoclonal anti-HA (ab1424, Abcam) or anti-Myc (M5546, Sigma) antibodies or no antibody for 2 h at 4 °C. Crude extract (100 μl) was added to the columns with the immobilized antibodies and incubated for 2 h at 4 °C. Washes were carried out with ice-cold wash buffer (20 mm HEPES-KOH, pH 7.7, 3 mm MgCl2, 150 mm KCl, 0.1% Nonidet P-40). Purified proteins were eluted into boiling SDS-PAGE loading buffer, boiled for 5 min, and centrifuged at 1000 × g for 7 min. The supernatant was removed, and 15 μl was loaded onto either 8 or 10% polyacrylamide gels.
6
Co-Immunoprecipitation of Myc-Tagged Proteins
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Anti-FLAG monoclonal antibody (F1804, Sigma-Aldrich), anti-Myc monoclonal antibody (m5546, Sigma-Aldrich), and immunoglobulin G (IgG) goat anti-rabbit polyclonal antibody (ab20272, 1:5000, Abcam) were applied to perform co-immunoprecipitation according to the standard protocol. Briefly, each 100-mm culture dish was cultured with 0.6 mL of 1× ice-cold cell lysis buffer containing 20 mM Tris-Cl (pH 7.5), 150 mM NaCl, 1 mM ethylene diamine tetraacetic acid (EDTA), 1 mM ethyleneglycol-bis (beta-aminoethylether)-N,N′-tetraacetic acid, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin, and 1 mM fresh phenylmethylsulfonyl fluoride on ice for 5 min to the transfected BMSCs, and BMSCs in the plate were transferred to the fresh microcentrifuge tubes. Cell lysates were then sonicated 4 times (5 s each time) on ice and centrifuged for 10 min at 4 °C. The primary anti-Myc antibody (2 μL) was supplemented to 200 μL of culture supernatant overnight at 4 °C. The supernatant was then added with 30 μL of 50% protein A-agarose beads (Upstate, Waltham, MA, USA), followed by 2 h culture at 4 °C. Beads were washed with 500 μL of 1× cell lysis buffer for 5 times. Proteins were then resuspended in 50 μL of SDS sample buffer and analyzed by Western blot analysis.
7
GFP-Trap Immunoprecipitation and Western Blot
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Cells were harvested after 24 h of plasmid transfection. Before collection, cells were washed 3 times with TBS and treated by ionomycin for 5 min at room temperature. Cells were lysed by buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, Roche protease inhibitor cocktail, and 1 mM EGTA at 4°C. After 1 h, cell supernatant was collected by centrifugation at 20,000g at 4°C for 30 min; 1 mL cell lysates were incubated with 15 μL GFP-Trap (ChromoTek) for 2 h at 4°C. The beads were washed 3 or 4 times by cold washing buffer composed of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Triton X-100, and 1 mM EGTA, followed by heating in 30 μL buffer containing SDS gel loading at 95°C for elution. Afterwards, eluted protein was loaded onto 11% SDS-PAGE gel and transferred to PVDF membranes for western blotting. The primary antibody of immunoblot analysis is anti-myc (M5546; Sigma-Aldrich, St. Louis) and anti-GFP (ab6556; Abcam, Cambridge, England). The protein–antibody complexes were detected using chemiluminescence.
8
Immunoprecipitation and Mass Spectrometry Analysis
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The immunoprecipitation experiment was performed as described in our previous publication [23 (link)] with the following modifications: 500 µg of protein extract from cMyc-A2AR284–410 expressing RAW 264.7 cells in RIPA buffer were diluted with PBS and supplemented with 100× Protease Inhibitor Cocktail (PIC, M221, VWR International, Radnor, PA, USA) and 100× Phenylmethylsulfonyl fluoride (PMSF) to 500 µL. 1 µg anti-cMyc antibody and its isotype control IgG1 (M5546, I5006, Sigma-Aldrich, St. Louis, MO, USA) were added to the lysates and incubated overnight with rotation at 4 °C. The total band protein content of the immuno-complexes formed with anti-cMyc antibody, were separated by SDS-PAGE and visualized by Coomassie Brillant Blue G250 staining, and the signal was detected with Fluorochem FC2 Imaging System (Alpha Innotech, Midland, ON, Canada). The whole band protein content was digested with trypsin and analyzed by mass spectrometry.
9
Immunoblotting of Protein Complexes
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The following antibodies were used: Peroxidase-Anti-Peroxidase (P1291; Sigma) to detect the protein A domain of TAP, anti-CBP against the Calmodulin Binding Protein domain of TAP (DAM1411288; Millipore), a monoclonal anti-HA antibody (H3663; Sigma), a monoclonal anti-c-MYC antibody (M5546; Sigma). Polyclonal antibodies against Taf9, Spt3 and Not5 were produced in rabbits (Elevage Scientifique des Dombes, France) and used at a 1:5000 dilution. The secondary antibodies were anti-Mouse-HRP (IgG-Peroxidase conjugate; A9044; Sigma) used at 1: 10 000 or anti-Rabbit-HRP (IgG-Peroxidase conjugate; A8275; Sigma) used at 1:10 000.
10
Coimmunoprecipitation of Myc and FLAG-tagged Proteins
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Coimmunoprecipitation was performed using anti-myc and anti-FLAG antibodies, as well as an anti-FLAG monoclonal antibody (F1804, Sigma), anti-myc monoclonal antibody (M5546, Sigma), and IgG goat anti-rabbit polyclonal antibody (ab20272, 1:5000, Abcam, UK). Then, transduced BMSCs were lysed on ice for 5 min. Then, the cell lysate was sonicated 4 times on ice (5 s each) and centrifuged at 4 °C for 10 min to harvest the supernatant (200 µl), which was incubated with the primary anti-myc antibody (2 µl) at 4 °C overnight. Next, the solution was incubated with 50% Protein A-agarose was at 4 °C for 2 h. The beads were washed five times with 500 µl of 1 × cell lysate. The protein was suspended in 50 µl of SDS sample buffer and analyzed by Western blotting.
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