Whole-cell lysates from sporulating cells (induced by resuspension) were prepared as described previously (Doan et al., 2009 (link)). Samples were heated for 10 min at 50°C prior to loading. Equivalent loading was based on OD600 at the time of harvest. Proteins were separated by SDS-PAGE on 12.5% polyacrylamide gels, electroblotted onto Immobilon-P membranes (Millipore) and blocked in 5% nonfat milk in phosphate-buffered saline (PBS)-0.5% Tween-20. The blocked membranes were probed with anti-SpoIID (1:10,000) (Doan & Rudner, 2007 (link)), anti-SpoIIQ (1:10,000) (Doan et al., 2005 (link)), anti-SpoIIIAH (1:10,000) (Doan et al., 2005 (link)), anti-SpoIIP (1:10,000) (Morlot et al., 2010 (link)), anti-σA (1:10,000) (Fujita, 2000 (link)), anti-EzrA (1:10,000) (Levin et al., 1999 (link)), anti-His (Genscript) (1:4000), anti-mCherry (1:10,000), diluted into 3% BSA in 1X PBS-0.05% Tween-20. Primary antibodies were detected using horseradish peroxidase-conjugated goat, anti-rabbit IgG (1:20,000, BioRad) and the Western Lightning reagent kit as described by the manufacturer (PerkinElmer).
Anti his
Manufactured by GenScript
Sourced in China
Anti-His is a laboratory equipment used for the purification and detection of histidine-tagged proteins. It enables the specific capture and isolation of recombinant proteins tagged with a histidine sequence, facilitating their subsequent analysis and downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p membrane, by Merck Group (3 mentions)
Horseradish peroxidase conjugated anti mouse or anti rabbit antibodies, by Bio-Rad (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti ha, by Merck Group (2 mentions)
Horseradish peroxidase conjugated goat anti rabbit or anti mouse igg, by Bio-Rad (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Bio-Rad (1 mentions)
Western lightning reagent kit, by PerkinElmer (1 mentions)
Amylose resin, by New England Biolabs (1 mentions)
Glutathione sepharose bead, by GE Healthcare (1 mentions)
Bca protein assay, by Beyotime (1 mentions)
Protein a agarose beads, by GE Healthcare (1 mentions)
Anti gfp, by GenScript (1 mentions)
Anti fibronectin, by Merck Group (1 mentions)
Anti e cadherin, by BD (1 mentions)
Anti gst, by GenScript (1 mentions)
Odyssey near infrared fluorescence scanner, by LI COR (1 mentions)
Hybond ecl transfer membrane, by GE Healthcare (1 mentions)
Ab16046, by Santa Cruz Biotechnology (1 mentions)
Ab16046, by Merck Group (1 mentions)
15 protocols using anti his
1
Western Blot Analysis of Sporulating Cells
2
Western Blot Analysis of Sporulating Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell lysates from sporulating cells were prepared as previously described [36 (link)]. Samples were heated for 15 min at 50°C prior to loading. Equivalent loading was based on OD600 at the time of harvest. Samples were separated on a 12.5% polyacrylamide gel and transferred to a PVDF membrane. Membranes were blocked in 5% non-fat milk with 0.5% Tween-20 for 1 h. Blocked membranes were probed with anti-GFP (1:5000) (BioRad), anti-His (1:4000) (Genscript), anti-SpoIIIAG [34 (link)] or anti-FtsZ [38 (link)] primary antibodies diluted into PBS with 5% non-fat milk (w/v) with 0.05% Tween-20 at 4°C overnight. Primary antibodies were detected with horseradish-peroxidase conjugated anti-mouse or anti-rabbit antibodies (BioRad) and detected with Western Lightning ECL reagent as described by the manufacturer.
3
Purification and Characterization of BISP Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The OsRLCK185–MBP–His, BISP–GST–His and BISP(26–124)–GST–His constructs were expressed in E. coli BL21 (DE3) cells. GST-tagged or MBP-tagged recombinant proteins were purified using Glutathione Sepharose beads (GE Healthcare, 17075601) and Amylose Resin (New England Biolabs, E8022S), respectively, according to the manufacturer’s instructions. Protein concentrations were determined by BCA protein assay (Beyotime, P0010S).
For the BISP antibody specificity analysis, the purified BISP–GST–His protein and total protein from whole insect and a twofold dilution series were separated on SDS–PAGE gels. BISP–GST–His was detected in immunoblots using anti-BISP and anti-His (GenScript, A00186; 1:2,000) antibodies, respectively. Ponceau S solution was used to stain the PVDF membrane and used as the total BPH protein loading control.
For the BISP antibody specificity analysis, the purified BISP–GST–His protein and total protein from whole insect and a twofold dilution series were separated on SDS–PAGE gels. BISP–GST–His was detected in immunoblots using anti-BISP and anti-His (GenScript, A00186; 1:2,000) antibodies, respectively. Ponceau S solution was used to stain the PVDF membrane and used as the total BPH protein loading control.
4
Immunoprecipitation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunoprecipitation, the cells were washed by cold PBS twice before being lysed in IP lysis buffer supplemented with protease and phosphatase inhibitors. Total protein lysate (2 mg) was used for each immunoprecipitation using specific antibody, Protein A agarose beads (GE Healthcare Uppsala, Sweden) were added to the cells and incubated overnight 4°C. Washed precipitated proteins were analyzed by western blot. The immunoprecipitation (IP), western blot and GST pull-down assays used in this study have been described previously in detail [23 (link)]. The samples were incubated with anti-E-cadherin (BD), anti-PAK5 (R&D), anti-GATA1/-Myc (Santa Cruz), anti-pGATA1 S161 (Bioss), anti-GFP (Genscript), anti-fibronectin (Sigma), anti-Flag (Shang Hai Ruixing), anti-His (Genscript), anti-GAPDH (Kang Chen, as a loading control) antibodies.
5
Comprehensive Mitochondrial Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
S. pombe whole cell extracts were prepared by alkaline extraction (58 (link)) or by breaking cells with glass beads using a FastPre-24 bead beater (MP Biomedicals) (59 (link)). Mitochondrial protein extracts were prepared as described above. Proteins were resolved by electrophoresis on SDS-PAGE, and the separated protein bands were transferred electrophoretically to a Hybond ECL transfer membrane (GE healthcare). Blots were probed with anti-Cox1 (1:1000), anti-Cox2 (1:1000), anti-Cox3 (1:400), anti-Cox4 (1:1000) and anti-Cob1 (1:500), anti-Trz2 (1:1000), anti-Sla1 (1:5000), anti-HSP60 (1:1000; Sangon Biotech), anti-FLAG (1:000; Sigma), anti-CBP (1:1000; GenScript), anti-HA (1:2000; Sigma-Aldrich), anti-c-Myc (1:2000; Affinity Biotech), anti-His (1:2000; GenScript) and anti-GST (1:2000; GenScript) Abs. Secondary Abs used were IRDye 800CW conjugated goat anti-rabbit or anti-mouse Abs (LI-COR Biosciences). Bands were detected using an Odyssey near-infrared fluorescence scanner (LI-COR Biosciences).
+ Expand
S. pombe whole cell extracts were prepared by alkaline extraction (58 (link)) or by breaking cells with glass beads using a FastPre-24 bead beater (MP Biomedicals) (59 (link)). Mitochondrial protein extracts were prepared as described above. Proteins were resolved by electrophoresis on SDS-PAGE, and the separated protein bands were transferred electrophoretically to a Hybond ECL transfer membrane (GE healthcare). Blots were probed with anti-Cox1 (1:1000), anti-Cox2 (1:1000), anti-Cox3 (1:400), anti-Cox4 (1:1000) and anti-Cob1 (1:500), anti-Trz2 (1:1000), anti-Sla1 (1:5000), anti-HSP60 (1:1000; Sangon Biotech), anti-FLAG (1:000; Sigma), anti-CBP (1:1000; GenScript), anti-HA (1:2000; Sigma-Aldrich), anti-c-Myc (1:2000; Affinity Biotech), anti-His (1:2000; GenScript) and anti-GST (1:2000; GenScript) Abs. Secondary Abs used were IRDye 800CW conjugated goat anti-rabbit or anti-mouse Abs (LI-COR Biosciences). Bands were detected using an Odyssey near-infrared fluorescence scanner (LI-COR Biosciences).
6
Co-Immunoprecipitation Assay Protocol
7
Probing Protein-Protein Interactions with Pull-Down Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pull-down assays were performed as described previously with minor modifications (Wang et al., 2018 (link)). pCold-TF-phyA-N (His-TF-phyA-N), pCold-TF-phyA-C (His-TF-phyA-C), pCold-TF-phyB-N (His-TF-phyB-N), and pCold-TF-phyB-C (His-TF-phyB-C) constructs were made previously (Wei et al., 2021 (link)). The DNA fragment of BIN2 was cloned into pGEX-4T-1 (GE Healthcare Life Sciences, United States). His-TF-phyA-N, His-TF-phyA-C, His-TF-phyB-N, His-TF-phyB-C and GST-BIN2 fusion proteins were expressed in E. coli (Rosetta). For GST pull-down assays, MagneGST™ Glutathione Particles (Promega, United States, V8611) were used. Prey proteins were detected with anti-His (GenScript, China, A00186).
8
Plasmid Transfection and Pulldown
9
Antibody-based Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The antibodies and chemicals used in the study were anti-Flag (Sigma, F1804 and Cell Signal Technology, CST 14793), anti-His (Genscript, A00186), anti-phosphoseine (Santa Cruz Biotechnology, Santa sc-81516), anti-PLK1 (Cell Signal Technology, CST 4513 and Santa, sc-17783), anti-UHRF1 (Proteintech, 21402-1-AP and Santa, sc-373750), anti-DNMT1 (Abclonal, A5495), anti-p21 (CST, 2947), anti-p16 (CST, 18769), anti-HA (CST, 3724), anti-5-Methylcytosine (CST, 28692), anti-Phospho-Histone H3 (Ser 10) (Proteintech, 66863-1) and anti-β-actin (Abclonal, AC004). Cycloheximide (CHX) was purchased from CST and MG132 and BI6727 were purchased from Selleck.
10
Immunoblot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunoblot analysis was performed as described previously (52 (link)). Briefly, 1 mL of culture was collected and resuspended in lysis buffer (20 mM Tris pH 7.0, 10 mM MgCl2 and 1mM EDTA, 1 mg/mL lysozyme, 10 μg/mL DNase I, 100 μg/mL RNase A, 1 mM PMSF) to a final OD600 of 10 for equivalent loading. The cells were incubated at 37 °C for 15 min followed by addition of an equal volume of Laemelli sample buffer (0.25 M Tris pH 6.8, 4% SDS, 20% glycerol, 10 mM EDTA) containing 10% β-mercaptoethanol. Samples were heated for 15 min at 65 °C prior to loading. Proteins were separated by SDS-PAGE on 12.5% polyacrylamide gels, electroblotted onto Immobilon-P membranes (Millipore) and blocked in 5% nonfat milk in phosphate-buffered saline (PBS) with 0.5% Tween-20. The blocked membranes were probed with anti-SigA (1:10,000) (53 (link)), anti-His (1:4,000) (GenScript), anti-GFP (1:10,000) (54 (link)) antibodies diluted into 3% BSA in PBS with 0.05% Tween-20. Primary antibodies were detected using horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (BioRad) and the Super Signal chemiluminescence reagent as described by the manufacturer (Pierce). Signal was detected using a Bio-Techne FluorChem R System.
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!