Prokaryotic expression plasmids fused with FLAG or GST tag were constructed including FLAG-HEATR5B-881aa, GST-JMJD5(-WT), GST-JMJD5-S361A. These plasmids were transformed into E. coli competent cell BL21 (TaKaRa, Liaoning, China) and protein expression was induced by 0.8 mM IPTG (Solarbio, Beijing, China) at 25 °C and 200 rpm for 6 h. Then, cells were lysed, sonicated, and centrifuged. The proteins were purified using the BeyoMag™ anti-Flag Magnetic Beads and BeyoGold™ GST-tag Purification Resin (Beyotime, Shanghai, China) according to the manufacturer’s procedure. For GST pull-down assay, purified FLAG-HEATR5B-881aa was incubated with purification resin coupled with GST-JMJD5 protein at 4 °C overnight. Then protein complexes were eluted by Elution Buffer and then subjected to SDS-PAGE and analyzed by western blot assays.
Beyomag anti flag magnetic beads
Manufactured by Beyotime
Sourced in China
BeyoMag™ anti-Flag Magnetic Beads are monodisperse superparamagnetic beads conjugated with anti-Flag antibodies. They are designed for efficient immunoprecipitation and purification of Flag-tagged proteins from various samples.
Automatically generated - may contain errors
Lab products found in correlation
Beyogold gst tag purification resin, by Beyotime (2 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Solarbio (2 mentions)
Flag peptide, by Beyotime (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Silver staining, by Beyotime (1 mentions)
Gel imaging system, by GE Healthcare (1 mentions)
Quickblock blocking buffer, by Beyotime (1 mentions)
Hrp labeled goat anti mouse secondary antibody, by Proteintech (1 mentions)
3xflag peptide, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Fast silver stain kit, by Beyotime (1 mentions)
Sds page sample loading buffer, by Beyotime (1 mentions)
Co ip lysis buffer, by Beyotime (1 mentions)
6 protocols using beyomag anti flag magnetic beads
1
Purification and interaction of HEATR5B and JMJD5
2
Immunoprecipitation and Mass Spectrometry Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following a previously described method [23 (link)], whole protein lysates were incubated with control IgG and 20 μl protein A/G plus-agarose (Santa Cruz Biotechnology, USA) at 4 ℃ for 30 min. The lysates were then centrifuged to remove nonspecific bound proteins. Subsequently, the lysates were co-incubated with the specific primary antibody for 1 h at 4 ℃, followed by co-incubation with 20 μl protein A/G plus agarose overnight at 4 ℃. The immune complexes were collected by centrifugation, washed five times, and subjected to immunoblot analysis.
For silver staining and immunoprecipitation-mass spectrometry (IP-MS), Flag-Sox17 was expressed in vascular endothelial cells, and whole protein lysates were extracted. The lysates were then co-incubated with BeyoMag™ anti-Flag magnetic beads (Beyotime, China) and eluted using 3 × Flag peptide (Beyotime) to remove IgG light and heavy chains. The eluate was separated using SDS-PAGE for mass spectrometry (MS; Thermo Fisher Scientific, USA) and silver staining (Beyotime) according to the manufacturer’s instructions.
For silver staining and immunoprecipitation-mass spectrometry (IP-MS), Flag-Sox17 was expressed in vascular endothelial cells, and whole protein lysates were extracted. The lysates were then co-incubated with BeyoMag™ anti-Flag magnetic beads (Beyotime, China) and eluted using 3 × Flag peptide (Beyotime) to remove IgG light and heavy chains. The eluate was separated using SDS-PAGE for mass spectrometry (MS; Thermo Fisher Scientific, USA) and silver staining (Beyotime) according to the manufacturer’s instructions.
3
Co-Immunoprecipitation of LcMFN2 and LcMAVS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T cells seeded in 10-cm plates (1 × 107 cells/well) were co-transfected with 5 μg FLAG-tagged LcMFN2 and 5 μg His-tagged LcMAVS or empty vector. After 24 h, cells were lysed with prechilled lysis buffer (50 mmol/L Tris HCL, pH 7.4, 150 mmol/L NaCl, 1 mmol/L EDTA, 1% Triton-X-100, and 1 mmol/L PMSF). Proteins were precipitated using BeyoMag™ Anti-Flag Magnetic Beads (Beyotime) at 4 °C for 6 h. Following three times washing with TBS/T, the immunocomplex was eluted with 3 X Flag Peptide (sigma). For immunoblotting, denatured proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electro-imprinted on 0.2 μmol/L PVDF membrane (Millipore, Bedford, MA) using a micro-trans-Blot cell system (Bio-Rad), and then blocked with Quick block blocking buffer (Beyotime). The membrane was incubated overnight at 4 °C with primary antibodies against FLAG-Tag (MBL, M185-3L, 1:10,000 dilution in TBS/T containing 1% non-fat dry milk) or His-Tag (1:3000 dilution in TBS/T containing 1% non-fat dry milk), followed by washing three times with TBS/T. Then, the membrane was probed with the HRP-labeled goat anti-mouse secondary antibody (Proteintech, SA0001-1, 1:10,000). Signals were detected using an ECL chemiluminescent system captured by a gel imaging system (GE Healthcare).
4
Immunoprecipitation-Mass Spectrometry for Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The entire experimental procedure for the Immunoprecipitation-mass spectrometry (IP-MS) analysis was referenced from the previous method with some modifications (Cao and Yan, 2016 (link)). Wild-type callus of M. truncatula was obtained via plant tissue culture. The ABI4-3302-3FLAG expression vector, constructed in a similar way to the aforementioned vectors, was transformed into callus by an Agrobacterium-mediated method for transient expression of ABI4 protein. In addition, the 3302-3FLAG expression vector was also transformed into the callus as a control.
After three days of dark incubation, the total protein of the callus was extracted by grinding in PBS buffer (pH = 7.4). The protein samples were purified using BeyoMag™ Anti-Flag Magnetic Beads (Beyotime, China). The end products obtained by elution were subjected to electrophoretic analysis using 10% SDS-PAGE gel. When bands were observed on the silver-stained gel treated with the Fast Silver Stain Kit (Beyotime, China), the gel block was sent to Bio for mass spectrometry analysis (Biomarker, China). The candidate proteins that interacted with the target protein were obtained by subtracting the protein in the control group from the experimental group results.
After three days of dark incubation, the total protein of the callus was extracted by grinding in PBS buffer (pH = 7.4). The protein samples were purified using BeyoMag™ Anti-Flag Magnetic Beads (Beyotime, China). The end products obtained by elution were subjected to electrophoretic analysis using 10% SDS-PAGE gel. When bands were observed on the silver-stained gel treated with the Fast Silver Stain Kit (Beyotime, China), the gel block was sent to Bio for mass spectrometry analysis (Biomarker, China). The candidate proteins that interacted with the target protein were obtained by subtracting the protein in the control group from the experimental group results.
5
Flag-tagged Protein Pulldown Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For FLAG pulldown assays, OCI-AML3 cells stably expressing FLAG-GFP, FLAG-RPS2, or FLAG-RPS2 (C222S) were treated with DMSO or 5 μmol/L HEL for 48 h. Cells were collected, washed with ice-cold PBS, and lysed by probe sonication in Co-IP lysis buffer (#P0013, Beyotime). After centrifugation at 12,000 × g for 10 min at 4 °C, the supernatant was incubated with BeyoMag Anti-Flag Magnetic Beads (#P2115, Beyotime), washed twice with cold TBS (#ST661, Beyotime), and the beads were added to SDS-PAGE sample loading buffer (#P0015, Beyotime) and boiled at 95 °C for 5 min. The supernatants were used for Western blot analyses.
For MDM2 pulldown assays, OCI-AML3 cells were treated with DMSO or 5 μmol/L HEL for 24 h, washed with cold PBS, and lysed with Co-IP lysis buffer. MDM2 was precipitated using protein A/G magnetic beads according to the manufacturer’s instructions; PBS was used for the washes. A specific isotype control antibody served as the control. The amounts of MDM2, p53, and RPL11 in the pulldown fractions were analyzed by Western blot analysis.
For statistical analyses, the results of each biological replicate were set to a 1.0-fold ratio.
For MDM2 pulldown assays, OCI-AML3 cells were treated with DMSO or 5 μmol/L HEL for 24 h, washed with cold PBS, and lysed with Co-IP lysis buffer. MDM2 was precipitated using protein A/G magnetic beads according to the manufacturer’s instructions; PBS was used for the washes. A specific isotype control antibody served as the control. The amounts of MDM2, p53, and RPL11 in the pulldown fractions were analyzed by Western blot analysis.
For statistical analyses, the results of each biological replicate were set to a 1.0-fold ratio.
6
Affinity Purification of Recombinant Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Prokaryotic expression plasmids fused with FLAG or GST tag were constructed including FLAG-CDK12, GST-MBNL1 (WT), GST-MBNL1-T6A (mut). These plasmids were transformed into Escherichia coli competent cell BL21 (Takara, Kyoto, Japan) and protein expression was induced by 1 mM IPTG (Solarbio, Beijing, China) at 25 °C and 200 rpm for 6 h. Then, cells were lysed, sonicated, and centrifuged. The proteins were purified using the BeyoMag™ anti-Flag Magnetic Beads and BeyoGold™ GST-tag Purification Resin (Beyotime Institute of Biotechnology, Jiangsu, China) according to the manufacturer’s procedure. For GST pull-down assay, purified FLAG-CDK12 was incubated with purification resin coupled with GST-MBNL1 protein at 4 °C overnight. Then protein complexes were eluted by Elution Buffer and then subjected to SDS-PAGE and analyzed by western blot.
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!