Total proteins were extracted from about two gram of Arabidopsis seedlings in cold IP buffer (50-mM Tris-Cl pH 7.6, 150-mM NaCl, 5-mM MgCl2, 10% (v/v) glycerol, 0.1% (v/v) NP-40, 0.5-mM DTT, and 1% protease inhibitor cocktail (sigma)). After cellular debris was removed by centrifugation, the supernatant was incubated with anti-14-3-3 antibody (ARS-AS12 2119, Agrisera) preincubated with Dynabeads (10003D, Invitrogen) or anti-GFP-Trap Magnetic Agarose (GTMA-20, Chrome Tek) at 4 °C for 3 h, and then the beads were washed for four times with IP buffer followed by two times wash with 1x PBS. The immunoprecipitated proteins were analyzed by Western blotting using anti-14-3-3 (ARS-AS12 2119, Agrisera), anti-GFP (D110008, Sangon Biotech) and anti-Actin (D110007, Sangon Biotech) antibodies.
Anti actin
Manufactured by Sangon
Sourced in United States
Anti-Actin is a laboratory antibody used to detect and quantify the presence of actin, a ubiquitous cytoskeletal protein found in eukaryotic cells. It is a widely used tool for research purposes.
Automatically generated - may contain errors
Lab products found in correlation
Dynabead, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Surepage, by GenScript (1 mentions)
Anti h3, by Proteintech (1 mentions)
Anti flag, by Merck Group (1 mentions)
Anti ha, by Merck Group (1 mentions)
Anti gfp, by Abcam (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Ecl prime western blot detection reagent, by GE Healthcare (1 mentions)
Anti c myc, by Roche (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti ha, by Roche (1 mentions)
Horseradish peroxidase conjugated goat anti mouse igg, by Merck Group (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Anti gfp, by Roche (1 mentions)
Anti p stat6, by Abcam (1 mentions)
Anti stat6, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca protein assay, by Beyotime (1 mentions)
4 protocols using anti actin
1
Immunoprecipitation of 14-3-3 Proteins
2
Protein Detection and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were resolved on 4–12% protein gels (GenScript, SurePAGE, Cat. no. M00653) and detected by anti-GFP (Abcam, cat. no. ab290, Lot#GR3196305-1), anti-HA (Sigma-Aldrich, cat. no. H6533), anti-FLAG (Sigma-Aldrich, cat. no. A8592), anti-ACTIN (Sangon Biotech, cat. no. D191048) and anti-H3 (Proteintech, cat. no. 17168-1-AP). The intensities of blotting signals were quantified by using ImageJ software.
3
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare, 10600003). Membranes were incubated with primary antibodies and secondary antibodies sequentially. Primary antibodies we used include anti-GFP (Roche, 11814460001, 1:5000 dilution), anti-c-myc (Roche, 11667203001, 1:2000 dilution), anti-Flag M2 (Sigma, F1804, 1:5000 dilution), anti-HA (Roche, 11666606001, 1:5000 dilution), anti-actin (Sangon, D191048, 1:5000 dilution) and anti-α-tubulin (Sigma, T5168, 1:5000 dilution). Horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma, A4416, 1:5000 dilution) was used as the secondary antibody. ECL prime western blot detection reagent (GE Healthcare, RPN2232) was added to the membranes for chemiluminescence detection using Image Lab (Bio-Rad).
4
Comprehensive Western Blot Analysis of Protein Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The western blot assay was performed to detect the protein expression. The total proteins were obtained by RIPA buffer (CST, USA), and the concentration was measured by BCA protein assay (Beyotime, China). An equal amount of proteins (80 μg) were electrophoresed through sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE). Then protein strips were transferred to the PVDF membranes (Millipore, USA). The membrane was blocked with 5% nonfat milk. And then incubated with the corresponding primary antibody overnight, including anti-STAT6 (Abcam, USA), anti-p-STAT6 (Abcam, USA), anti-p-BCR-ABL (CST, USA), anti-Caspase-3 (CST, USA), anti-PARP (CST, USA), anti-p27 (CST, USA), anti-p21 (Bimake, USA), anti-Cdk1/Cdc2 (Bimake, USA), anti-Cdc25c (Bimake, USA), anti-PCNA (Bimake, USA), anti-c-Myc (CST, USA), anti-Jak1 (CST, USA), anti-Jak2 (CST, USA), anti-p-Jak2 (CST, USA), anti-Jak3 (CST, USA), anti-Actin (Sangon Biotech, China). Then the membrane was incubated with the corresponding secondary antibody. The protein signal was developed with Chemistar™ high-sig ECL Western Blot Substrate (Tanon, China).
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!