Total protein was extracted from 10 females in RIPA buffer (150 mM NaCl, 1.0% IGEPAL, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl pH 8.0) plus protease inhibitors (Roche Applied Science). Samples were denatured for 5 min at 100°C, electrophoresed on 8% SDS-PAGE gels, transferred onto nitrocellulose membranes (Roche Applied Science) and immunodetected following standard procedures. Membranes were incubated with primary anti-GFP antibody (1 h, 1:1000, Roche Applied Science) followed by horseradish peroxidase (HRP)-conjugated anti-mouse-IgG secondary antibody (1 h, 1:3000, Sigma-Aldrich). Loading control was anti-tubulin (overnight, 1:5000, Sigma-Aldrich) followed by incubation with HRP-conjugated anti-mouse-IgG secondary antibody (1 h, 1:3000, Sigma-Aldrich). Bands were detected using ECL Western Blotting Substrate (Pierce). Images were obtained using the camera system ImageQuant LAS 4000 (GE Healthcare Australia Pty Ltd, Rydalmere, NSW, Australia).
Anti gfp primary antibody
Manufactured by Roche
Sourced in Switzerland
The Anti-GFP primary antibody is a laboratory reagent used to detect and study the green fluorescent protein (GFP) in various biological samples. It is a specific antibody that binds to the GFP protein, enabling its visualization and analysis through various techniques such as Western blotting, immunohistochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Roche (2 mentions)
Nitrocellulose membrane, by Roche (1 mentions)
Hrp conjugated anti mouse igg secondary antibody, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated anti mouse igg secondary antibody, by Merck Group (1 mentions)
Anti tubulin, by Merck Group (1 mentions)
Alexa fluor 568 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Enhanced chemifluorescence, by Cytiva (1 mentions)
Storm phosphoimager, by GE Healthcare (1 mentions)
Imagequant, by GE Healthcare (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
Poly horseradish peroxidase poly hrp conjugated goat anti mouse antibody, by Thermo Fisher Scientific (1 mentions)
Ponceau s, by Merck Group (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
6 protocols using anti gfp primary antibody
1
GFP Protein Detection via Western Blot
2
Visualization of IRE1α Foci in HEK293 Cells
3
Quantifying Protein Abundance in Cellular Fractions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For all protein experiments, protein extracts were prepared as previously described (25 (link), 26 (link)). Briefly, the WT (H99) and the Ali1-GFP (CLT7) strains were incubated for 18 h at 30°C with 150 rpm shaking in YPD medium. Cells were pelleted, flash frozen on dry ice, and lysed by bead beating. The crude lysate was cleared by centrifugation at 2,500 × g at 4°C for 5 min, and the supernatant (total cell lysate) was transferred to a new tube. Total cell lysate protein concentrations were measured using a bicinchoninic acid assay (BCA).
To determine the relative abundance of Ali1 in different cellular fractions, the WT (H99) and Ali1-GFP (CLT7) strains were incubated and lysed as described above. Total cell lysates (T) were separated by ultracentrifugation at 30,000 × g for 1 h at 4°C (27 (link)). The soluble fraction (S) was transferred to a new tube, and the insoluble pellet (I) was resuspended in an equivalent volume of lysis buffer containing 1% Triton X‐100. All samples were normalized by total protein concentration. Western blots were performed as described previously using an anti‐GFP primary antibody (1/5,000 dilution; Roche), followed by an anti‐mouse peroxidase‐conjugated secondary antibody (1/25,000 dilution; Jackson Laboratory). Proteins were detected by enhanced chemiluminescence (ECL Prime Western blotting detection reagent; GE Healthcare).
To determine the relative abundance of Ali1 in different cellular fractions, the WT (H99) and Ali1-GFP (CLT7) strains were incubated and lysed as described above. Total cell lysates (T) were separated by ultracentrifugation at 30,000 × g for 1 h at 4°C (27 (link)). The soluble fraction (S) was transferred to a new tube, and the insoluble pellet (I) was resuspended in an equivalent volume of lysis buffer containing 1% Triton X‐100. All samples were normalized by total protein concentration. Western blots were performed as described previously using an anti‐GFP primary antibody (1/5,000 dilution; Roche), followed by an anti‐mouse peroxidase‐conjugated secondary antibody (1/25,000 dilution; Jackson Laboratory). Proteins were detected by enhanced chemiluminescence (ECL Prime Western blotting detection reagent; GE Healthcare).
4
Western Blot Detection of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed once with PBS and lysed in RIPA buffer (50 mM Tris pH 7.5, 0.1% SDS, 150 mM NaCl, 0.5% sodium deoxycholate, 1% Triton X-100, protease inhibitor [Roche]). Protein samples (∼40 μg) were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred to Immobilon P membranes (Millipore). Western blots were performed using anti-GFP primary antibody (Roche) for detecting YFP protein expression. The anti-Insig1 primary antibody was purchased from Biovision. Blots were treated by alkaline phosphatase secondary antibodies (Invitrogen) and developed using enhanced chemifluorescence according to the manufacturer's instructions (Amersham GE Healthcare). Signal was detected using a Storm Phosphoimager and quantitated with ImageQuant (GE Healthcare); or blots were processed with horseradish peroxidase-conjugated secondary antibodies and developed using Pierce ECL Western Blot Substrate (Amersham GE Healthcare), according to the manufacturer's instructions, and exposed to Biomax Light Films.
5
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
2 x 107 log-phase cells were resuspended in 100 μl H2O. An equal volume of 0.2 M NaOH was added to the cell suspension, and cells were incubated 5 min at room temperature. Samples were then centrifuged at 20,000 x g for 10 min at 4°C. Pellets were resuspended in SDS-lysis buffer (10 mM Tris-HCl, pH 6.8, 100 mM NaCl, 1% SDS, 1 mM EDTA, and 1 mM EGTA) containing protease inhibitors (leupeptin, pepstatin, PMSF, and aprotinin) for western blot analysis. Immunoblotting was carried out exactly as previously described (Hughes and Gottschling, 2012 (link)). Anti-GFP primary antibody was from Roche (Switzerland) (#11814460001), and secondary HRP-conjugated antibodies from Jackson Immunoresearch (West Grove, PA).
6
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected by centrifugation, washed serially with cold water and lysis buffer (50 mM Tri-HCl, 500 mM NaCl, pH 7.4, supplemented with protease inhibitors: 1 mM PMSF, 4 mM benzamidine hydrochloride, 2.5 mM EDTA, pH 8) then disrupted with glass beads66 (link). Lysates were treated with 1% Triton X-100 on ice for 30 min and then with cracking buffer (8 M Urea, 5% (w/v) SDS, 40 mM Tris-HCl pH 6.8, 0.1 mM EDTA, 0.4 mg/ml bromophenol blue) at 37 °C for 10 min followed by incubation at 95 °C for a further 10 min. For western blotting, proteins were separated by electrophoresis on 10% (w/v) NuPAGE Bis-Tris gels (Life Technologies) before transfer to nitrocellulose membrane (GE Healthcare). Protein loading was shown by staining with Ponceau S (Sigma). Immunodetection of GFP tagged proteins was with anti-GFP primary antibody (1:1000 dilution; Roche) and poly horseradish peroxidase (poly HRP) conjugated goat anti-mouse antibody (1:10000 dilution; Thermo Scientific). GFP tagged proteins were detected with an electrochemiluminescence HRP kit (Pierce) and imaged using a Chemidoc XRS (Bio-Rad). Protein band intensities were quantified with ImageJ software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!