4×107 cells of each line expressing a LAP-RAS mutant construct were lysed in 300 μL of 150mM KCl, 50mM HEPES pH 7.4, 1mM MgCl2 10% glycerol, and 0.3% NP-40 (lysis buffer) supplemented with 3μL phosphatase inhibitor cocktail 2 (Sigma-Aldrich) and 1μg/mL each of leupeptin, pepstatin, and chymostatin (Sigma-Aldrich) on ice for 10 minutes. Cells were centrifuged for 15min at 4C. Supernatant concentration was measured by Bradford assay and diluted to 9mg/mL protein in 300μL lysate. For a 5% input sample, 9 μL were withheld and mixed with 4× LDS sample buffer +2.5% β-mercaptoethanol. 4.83 μL protein A beads conjugated to Rabbit αGFP antibody (Thermo Fisher) were added to the remaining supernatant. Samples were incubated 16h at 4C. Beads were washed 5× with 200μL 200mM KCl, 50mM HEPES pH 7.4, 1mM MgCl2 10% glycerol, and 0.3% NP-40 (lysis buffer), then immunoblotted as described (61 (link)), probed 16h at 4°C with rabbit αRAF1 (Abcam) and mouse αGFP (Invitrogen).
Mouse α gfp
Manufactured by Thermo Fisher Scientific
Mouse α-GFP is a primary antibody that specifically recognizes and binds to the green fluorescent protein (GFP) derived from the jellyfish Aequorea victoria. This antibody can be used to detect and visualize GFP-tagged proteins in various experimental applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)
Leupeptin, by Merck Group (1 mentions)
Pepstatin, by Merck Group (1 mentions)
Rabbit αgfp antibody, by Thermo Fisher Scientific (1 mentions)
Chymostatin, by Merck Group (1 mentions)
Ecl detecting reagent, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (1 mentions)
Fluoromount, by Southern Biotech (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Rat α ha, by Roche (1 mentions)
Alexa fluor 633, by Thermo Fisher Scientific (1 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (1 mentions)
A11122, by Thermo Fisher Scientific (1 mentions)
A11120, by Thermo Fisher Scientific (1 mentions)
Rabbit α gfp, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
7 protocols using mouse α gfp
1
Immunoprecipitation of LAP-RAS Mutants
2
Protein Extraction and Immunoblot Analysis
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Total proteins were extracted from 100 mg of sample using extraction buffer (100 mM Tris-Cl pH8, 150 mM NaCl, 0.6% IGEPAL, 1 mM EDTA, 3 mM DTT with protease inhibitors, PMSF, leupeptin, aprotinin, pepstatin, antipain, chymostatin, Na2VO3, NaF, MG132, and MG115. Proteins were separated on a 10% polyacrylamide gel. Immunoblot analysis was carried out using mouse α-GFP (1:2000; Invitrogen) for TuMV GFP and rat α-HA (1:500) antibody for pCas13a. The antigens were detected by chemiluminescence using an ECL-detecting reagent (Thermo Scientific).
3
Immunostaining of Drosophila Ovaries
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Antibody stainings were performed on ovaries dissected in Drosophila Schneider medium (Greiner), fixated for 15 min in 4% paraformaldehyde (Sigma-Aldrich) in PBS, permeabilized for 1 h in 0.1% Triton X-100 (Invitrogen) in PBS, blocked for 30 min in 3% BSA and stained with first antibody overnight at 4°C and for 2 h at RT with the second antibody (Zobel and Bogdan, 2013 (link)). The following primary antibodies were used rabbit α-AbiA8 (1:1,000; this work), guinea pig α-WAVE (1:1,000; Bogdan et al., 2005 (link)), α-Dlar (1:500; DSHB), mouse α-GFP (1:1,000; Invitrogen). Secondary antibodies were Alexa Fluor 488– or 647–conjugated anti–rabbit, anti–guinea pig, and anti–mouse (Molecular Probes), all used at 1:1,000. F-actin was stained by either Alexa Fluor phalloidin 568 or Alexa Fluor phalloidin 488 (1:100; Invitrogen). Samples were mounted in Fluoromount (SouthernBiotech).
4
Whole-Mount Immunofluorescence Staining of Brains
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Whole-mount immunofluorescence staining was carried out as described (Laissue et al., 1999 (link); Vosshall et al., 2000 (link)). Initially brains were dissected in Ringer's solution (130 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 36 mM saccharose, 5 mM HEPES, [pH 7.3]) (Estes et al., 1996 (link)) and fixed in 4% PFA in PBS-T (PBS, 0.2–1% Triton-X). After washing with PBS-T brains were blocked with PBS-T, 5% normal goat serum (NGS). Primary antibodies were diluted in blocking solution or PBS-T and incubated at 4°C for 2–3 days. Secondary antibody incubation lasted 1–2 days. Brains were mounted in VectaShield (Vector Labs, Burlingame, CA). The following primary antibodies were used: rabbit α-GABA (1:500) (Sigma), mouse α-GFP (1:500) (Invitrogen). The following secondary antibodies were used: Alexa Fluor 488, goat anti-mouse (1:500); Alexa Fluor 546, goat anti-rabbit (1:500); (all IgG Invitrogen).
5
Comprehensive Western Blot Antibody Protocol
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Western blotting was performed as described [60 (link)]. Primary antibodies used were: mouse α-Myc 9E10 antibody (1:2000, Enzo Life Sciences); mouse α-FLAG M2 antibody (1:1000, Sigma-Aldrich); rabbit α-GFP TP401 antibody (1:5000, Acris Antibodies); mouse α-HA F7 (1:1000, Santa-Cruz) rat α-HA (1:750, Roche); mouse α-β-Tubulin (1:5000, Covance), mouse α-α-Tubulin (1:20,000; Sigma), mouse α-GFP (1:500; Molecular probe), mouse α-ubiquitin (1:1000; Santa Cruz) Fab α-pAbp (2.5 μg in 5 ml), α-eIF4G rabbit antibody (1 μg in 5 ml), rabbit α-Osk (1:1000) antibody was a gift from A. Ephrussi.
6
Immunohistochemical Labeling of Cellular Targets
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Immunohistochemistry was performed following standard procedures (Bainton et al., 2005 (link); Schwabe et al., 2005 (link)). The antibodies used in the study were rabbit α-PkaC1 (1:400, Pka-C1, RRID:AB_2568479 ; Lane and Kalderon, 1993 (link)), mouse α-PkaC1 (1:100, BD), mouse α-REPO (1:10, Developmental Studies Hybridoma Bank), mouse α-GFP (1:100, Molecular Probes), mouse α-Mega (1:100, R. Schuh), guinea pig α-dContactin (1:1000, M. Bhat), and rabbit α-RFP (1:100, US Biological). Fluorescent secondary antibodies were coupled to Cy3 (1:500, Jackson), Alexa Fluor 488 or Alexa Fluor 633 (1:500, Molecular Probes). Rat α-Moody β was generated in the lab (1:500; Bainton et al., 2005 (link); Schwabe et al., 2005 (link)).
7
Drosophila Gut Immunohistochemistry Protocol
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The Drosophila gastrointestinal tract was dissected into 4% paraformaldehyde in PLP buffer and fixed for 1 h at room temperature. The tissue washes were performed with PBT (1× PBS and 0.1% Triton X-100). Antibody incubations were performed in PBT supplemented with 3% BSA and 0.02% sodium azide. Primary antibodies incubated at room temperature for at least 4 h or overnight at 4°C; secondary antibody incubations were 2 h at room temperature. Fixed and stained tissue was whole mounted in Vectashield mounting medium containing DAPI from Vector Laboratories. Primary antibodies used in this study included mouse α-FK2 (polyubiquitin; 1:200; Enzo Life Sciences), rabbit α-GFP (1:5,000; A11122 ; Molecular Probes), mouse α-GFP (1:200; A11120 ; Molecular Probes), chicken α-GFP (1:1,000; Aves Labs Inc.), rabbit α-βGal (1:2,000; Cappel), rabbit α(phospho–histone-H3 (1:200; 06–570; EMD Millipore), rabbit α-H2AvD pS137 (1:200; 600–401-914; Rockland), and mouse α-βGal (1:20; 40-1a), mouse α-Armadillo (1:20; N2 7A1), mouse α-HP1 (C1A9), and mouse α-Prospero (1:100; MR1A; Developmental Studies Hybridoma Bank).
Superresolution images were obtained from a custom-build STED superresolution microscope currently reaching a resolution of ∼30 to 40 nm.
Superresolution images were obtained from a custom-build STED superresolution microscope currently reaching a resolution of ∼30 to 40 nm.
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