Total cell protein was extracted with extraction buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 10 mM DTT, 10 mM Na3VO4, 20 mM NaF, 50 mM β-glycerophosphate, 10% glycerol, 1 mM PMSF, protease inhibitor cocktail for plant cell extracts (Sigma-Aldrich, P 9599), 1% (v/v) NP-40) from 3-d.p.g. Arabidopsis seedlings of Col-0, bsl1;bsl3, bsl-quad and pTMM::BSL1–YFP. The extracted total cell protein was resolved on 10% SDS–PAGE, followed by immunoblotting with a primary antibody against phosphor-p42/44 MAP kinase (1:1,000; Cell Signaling Technology). Total protein staining (Ponceau S) was used to confirm equal loading for immunoblots.
Protease inhibitor cocktail for plant cell extracts
Manufactured by Merck Group
Protease inhibitor cocktail for plant cell extracts is a laboratory product designed to prevent the degradation of proteins in plant cell samples. It contains a mixture of compounds that inhibit the activity of various proteases, enzymes that can break down proteins. This product is intended to be added to plant cell extracts to maintain the integrity of the proteins present, facilitating downstream analysis and research.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using protease inhibitor cocktail for plant cell extracts
1
Arabidopsis Protein Extraction and Immunoblot
2
Arabidopsis Protein Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cell proteins were extracted with extraction buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 10 mM DTT, 10 mM Na3VO4, 20 mM NaF, 50 mM β-glycerophosphate, 10% glycerol, 1 mM PMSF, protease inhibitor cocktail for plant cell extracts (Sigma-Aldrich, P 9599), 1% (v/v) NP-40) from 5-dpg Arabidopsis seedlings of Col-0, bsl2;bsl3;bsu1, bsl1;bsl2;bsl3, TMMp::myr-BSL2-mRFP, and TMMp::BSL2-nls-mRFP. The extracted total cell proteins were resolved on 10% SDS–PAGE, followed by Immunoblot with the primary antibody against phosphor-p42/44 MAP kinase (1:1000, Cell Signaling Technology). Equal loading was indicated by immunoblot analysis with anti-MPK6 antibody.
3
Chromatin Immunoprecipitation from MM2d Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MM2d cells were harvested 4 days after subculture and fixed using ice-cold 1% formaldehyde in PBS and applying vacuum infiltration (three rounds of 6 min on/4 min off). The cross-linking was stopped by the addition of 0.125 M glycine, infiltrating for another 5 min. The grinded material was resuspended in Extraction Buffer (0.25 M sucrose, 10 mM Tris–HCl, pH 8.0, 10 mM MgCl2, 1% Triton X-100, 1 mM PMSF and protease inhibitor cocktail for plant cell extracts (Sigma)). Nuclei were pelleted by centrifugation, resuspended in Lysis Buffer (50 mM Tris–HCl, pH 8.0, 10 mM EDTA, 1% SDS, 1 mM PMSF and protease inhibitor cocktail) and disrupted by sonication in a Bioruptor Plus (Diagenode) for 30–45 cycles of 30 s on and 30 s off, at high power mode. One μg of soluble chromatin was employed per ChIP reaction, using the following antibodies: anti-H3K9me2 (Abcam ab1220, 3 μg), anti-H3K27me1 (Millipore 07-448, 1 μg), anti-total H3 (Abcam ab1791, 2 μg), or anti-rat IgG (Abcam ab6703, 2 μg) as a negative control. Immune complexes were recovered with 50 μl of protein G agarose beads (SCBT) and washed and eluted essentially as described (28 (link)).
4
Co-immunoprecipitation of GFP-tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For co-immunoprecipitation, 3–6 g of leaf material was ground to a fine powder in liquid nitrogen and resuspended in extraction buffer [50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 1 mM EDTA, 5 mM DTT, 1% IGEPAL CA630 (Sigma), 1× protease inhibitor cocktail for plant cell extracts (Sigma)] at a ratio of 2 mL buffer/1 g leaf tissue. The homogenized extract was centrifuged at 4°C/20000×g/20 min and the supernatant was passed through a double layer of Miracloth. GFP-tagged proteins were immunoprecipitated by adding 25 µL of GFP-Trap beads (Chromotek) followed by incubation on a rolling wheel at 4°C for 2 h. The beads were collected by centrifugation at 4°C/1000×g/2 min, resuspended in 1 mL extraction buffer and transferred to 1.5 mL tubes. The beads were washed by 3 further rounds of centrifugation (4°C/1000×g/1 min) followed by resuspension in 1 ml extraction buffer. To extract proteins, the beads were boiled for 5 min in 45 µL of 1× SDS sample buffer.
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!