Commercial antibodies used for western blotting analysis include: anti-Phospho-Akt (Ser473) (#3787, 1:1000 dilution), anti-Phospho-Akt Substrate (R××pS/pT) (#9614, 1:1000 dilution for WB and 1:250 dilution for IP), anti-USP14 (rabbit, #11931, 1:1000 dilution), anti-FLAG (#2368, 1:1000 dilution) were from Cell Signaling Technology. Anti-phosphoserine (#61-8100, 1:1000 dilution for WB and 1:250 dilution for IP) was from Invitrogen. Anti-Akt (sc-8312, 1:1000 dilution) and anti-USP14 (mouse, sc-393872, 1:1000 dilution) were from Santa Cruz Biotechnology. Anti-Myc (16286-1-AP, 1:1000 dilution), anti-HA (51064-2-AP, 1:1000 dilution), and anti-GAPDH (60004-1-Ig, 1:10000 dilution) were from Proteintech. Anti-Tubulin (PM054, 1: 10000 dilution) was from MBL. Recombinant active Akt protein (#14-276) was from Millipore. Mouse monoclonal anti-FLAG (F1804, 1:500 for IP), anti-Myc (A7470), and anti-HA Affinity Gel (E6779) were from Sigma-Aldrich. IU1 (S7134), MK2206 (S1078), AZD5363 (S8019), GDC0941(S1065), Wortmannin (S2758), U0126 (S1102), and MG132 (S2619) were from Selleckchem. Phospho-USP14 S432 antibodies were generated by Proteintech. Briefly, synthetic peptides corresponding to phosphorylated S432 epitope (N'-Cys-THQGRSSs(phospho)SGHYVSW) of USP14 were used to immunize rabbits and the antisera were affinity purified.
Anti myc 16 286 1 ap
Manufactured by Proteintech
Sourced in United States
The Anti-myc, 16,286–1-AP is a primary antibody that recognizes the c-Myc protein. It is designed for use in various immunological techniques, such as Western blotting, immunoprecipitation, and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Anti ha 51064 2 ap, by Proteintech (3 mentions)
Anti ha affinity gel, by Merck Group (1 mentions)
Anti flag, by Merck Group (1 mentions)
Mg132 s2619, by Selleck Chemicals (1 mentions)
Anti gapdh 60004 1 ig, by Proteintech (1 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
Anti phosphoserine, by Thermo Fisher Scientific (1 mentions)
Anti akt sc 8312, by Santa Cruz Biotechnology (1 mentions)
Anti flag, by Cell Signaling Technology (1 mentions)
Alexa fluor 647, by BioLegend (1 mentions)
Anti flag a8592, by Merck Group (1 mentions)
Anti cd8 ab4055, by Abcam (1 mentions)
Anti α tubulin antibody t6074, by Merck Group (1 mentions)
Cd8 gb114196, by Wuhan Servicebio Technology (1 mentions)
F4 80 gb113373, by Wuhan Servicebio Technology (1 mentions)
Lsm 800 microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Anti smad2 3, by Cell Signaling Technology (1 mentions)
Anti smad2 3 sc 133098, by Santa Cruz Biotechnology (1 mentions)
Anti flag 20543 1 ap, by Proteintech (1 mentions)
10 protocols using anti myc 16 286 1 ap
1
Antibody Immunoblotting Protocol
2
KLF13 Interaction with TBX5 Studied
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
293 T cells were transfected with wild-type KLF13 or variants and TBX5 plasmids using FuGene HD according to the manufacturer’s guidelines. Protein extracts were incubated overnight with anti-myc antibody (1:100, rabbit polyclonal anti-myc, 16,286–1-AP, Proteintech, USA) coupled with magnetic beads. Bound proteins were revealed with anti-TBX5 (1:1000, rabbit polyclonal anti-TBX5, 42–6500, Invitrogen, USA) antibody by Western blotting.
3
Multiparameter Immune Cell Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-Mo CD3e (2074540; 1:100 dilution) and anti-Mo CD8a (2075802; 1:100 dilution) antibodies were purchased from Invitrogen (CA, USA). Alexa Fluor® 647 (B319823; 1:100 dilution) was purchased from BioLegend (CA, USA). Anti-CD8 (ab4055; 1:500 dilution) was purchased from Abcam (Illinois, USA). Anti-RNF125 (A15166; 1:1,000 dilution) was purchased from ABclonal (Wuhan, China). Anti-α-tubulin antibody (T6074; 1:5000 dilution) and anti-Flag (A8592; 1:5000 dilution) antibody were purchased from Sigma–Aldrich (Missouri, USA). Anti-PD-L1 (17952-1-AP; 1:1,000 dilution) and anti-Myc (16286-1-AP; 1:1,000 dilution) antibodies were purchased from Proteintech (Wuhan, China). Anti-HA (14031S; 1:1,000 dilution) was purchased from Cell Signaling Technology (Danvers, USA). Anti-rabbit HRP-IgG (ZB-2301; 1:5000 dilution) and anti-mouse HRP-IgG (ZB-2305; 1:5000 dilution) secondary antibodies were purchased from ZSGB-BIO (Beijing, China). CD4 (GB11064; 1:1000 dilution), CD8 (GB114196; 1:1000 dilution) and F4/80 (GB113373; 1:1000 dilution) antibodies were purchased from Servicebio (WuHan, China).
4
Immunoprecipitation and Immunoblotting Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
IP and IB were conducted as previously described (Yan et al., 2018 (link)).
Antibodies used in IP and IB are as follows: anti-TEAD4 (12418-1-AP) and anti-Myc (16286-1-AP) antibodies were bought from Proteintech; anti-HA (390025) and anti-Flag (700002) antibodies were purchased from ZEN BIO; anti-Smad4 (46535S), pan-TEAD (13295S), anti-YAP (12395S), anti-TAZ (4883S), and anti-p300 (86377S) antibodies were bought from Cell Signaling Technology; and anti-GAPDH (sc-47724), anti-β-actin (sc-8432), and Anti-Smad2/3 (sc-133098) antibodies were purchased from Santa Cruz Biotechnology.
For immunofluorescence, cells were sequentially treated with 4% paraformaldehyde for 10 min, 0.5% Triton X-100 for 5 min, 0.5% bovine serum albumin for 30 min, the primary antibody overnight at 4°C, and finally the secondary antibody for 1 h in the dark. The nuclei were counterstained with DAPI (Sigma). Images were obtained with a confocal ZEISS LSM 800 microscope. Anti-Smad2/3 (Cell Signaling Technology), anti-HA (ZEN BIO), and anti-TEAD4 (Proteintech) antibodies were used for immunofluorescence.
Antibodies used in IP and IB are as follows: anti-TEAD4 (12418-1-AP) and anti-Myc (16286-1-AP) antibodies were bought from Proteintech; anti-HA (390025) and anti-Flag (700002) antibodies were purchased from ZEN BIO; anti-Smad4 (46535S), pan-TEAD (13295S), anti-YAP (12395S), anti-TAZ (4883S), and anti-p300 (86377S) antibodies were bought from Cell Signaling Technology; and anti-GAPDH (sc-47724), anti-β-actin (sc-8432), and Anti-Smad2/3 (sc-133098) antibodies were purchased from Santa Cruz Biotechnology.
For immunofluorescence, cells were sequentially treated with 4% paraformaldehyde for 10 min, 0.5% Triton X-100 for 5 min, 0.5% bovine serum albumin for 30 min, the primary antibody overnight at 4°C, and finally the secondary antibody for 1 h in the dark. The nuclei were counterstained with DAPI (Sigma). Images were obtained with a confocal ZEISS LSM 800 microscope. Anti-Smad2/3 (Cell Signaling Technology), anti-HA (ZEN BIO), and anti-TEAD4 (Proteintech) antibodies were used for immunofluorescence.
5
Antibody Procurement and Immunoprecipitation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Anti-Flag (20543-1-AP; 1:1,000), anti-HA (51064-2-AP; 1:1,000), anti-MYC (16286-1-AP; 1:2,000) and anti-USP49 (18066-1-AP; 1:1,000) antibodies were purchased from Proteintech. Anti-FBXO45 antibody (orb156851; 1:1,000) was purchased from Biorbyt Company. Anti-tubulin antibody (2128 S; 1:1,000) was purchased from Cell Signaling Technology. Peroxidase-conjugated anti-mouse secondary antibody (70-GAM007, 1:5,000) and peroxidase-conjugated anti-rabbit secondary antibody (70-GAR0072; 1:5,000) were purchased from MultiSciences Company. Immunoprecipitation kit-HA tag immunomagnetic beads (TB100028, 50 μl for IP) and immunoprecipitation kit-DYKDDDDK (Flag) tag immunomagnetic beads (TB101274, 50 μl for IP) were purchased from Sino Biological Company.
6
Immunoblotting Analysis of Transcription Factor Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
293 T cells were harvested at 48 h after transfection with wild-type KLF13 or its variants. The protein was extracted as previously described [14 (link)]. A total of 50 μg of the extract was subjected to 10% SDS-PAGE, transferred onto nitrocellulose membranes and blocked with skim milk (5%) in TBST (0.1%) at room temperature for 2 h, using gentle agitation during the process. Then, the membranes were incubated with the primary antibody (myc: 1:1000, rabbit polyclonal anti-myc, 16,286–1-AP, Proteintech, USA; GAPDH: 1:1000, rabbit monoclonal anti-GAPDH, ab181602, Abcam, USA) overnight at a temperature of 4 °C. Following incubation with the primary antibody, horseradish peroxidase-conjugated secondary antibody (1:1000, 111–005-003, Jackson ImmunoResearch, USA) and Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA) were used to visualize chemiluminescent immunodetection.
7
Co-Immunoprecipitation of Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in immunoprecipitation lysis buffer (Thermo Fisher Scientific) supplemented with a complete protease inhibitor cocktail (Roche). Co-IP was performed using the SureBeads Protein A magnetic beads (Bio-Rad Laboratories) according to the manufacturer’s instructions. SureBeads were conjugated with 1 μg antibodies for 15 min. The following antibodies were used; anti-GFP (SC-8334) from Santa Cruz Biotechnology, anti-HA (51064-2-AP) and anti-MYC (16286-1-AP) from Proteintech, anti-STING (CST#13647) from Cell Signaling Technology, anti-MAVS (A300-782A) was purchased from Fortis Life Science and anti-LC3 (NB100-2220) from Novus. After antibody conjugation, 500 μg cell lysate was added, and incubated overnight at 4 °C. Immunoprecipitates were washed with 0.1% PBS/Tw three times, and proteins were eluted in SDS sample buffer and analyzed by immunoblot anaylsis to detect endogenous interactions between proteins. To examine ubiquitinated Parkin, lysed cells were incubated with magnetic beads which were conjugated with Parkin antibody (CST#4211) for 24 h and then immunoprecipitates were analyzed by immunoblot assay using phospho-ubiquitin (CST#62802).
8
Dissecting EGFR Signaling Mechanisms
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tamoxifen and MG132 were purchased from Sigma. Cycloheximide (CHX) was purchased from Amresco. LDN-57444 was purchased from Calbiochem. Fulvestrant and 17β-estradiol (E2) were purchased from Selleck. Antibodies used in immunoblotting: UCH-L1 (No.13179, 1:1000), ERα (No.8644, 1:1000), EGFR (No.4267, 1:1000), HA (No.3724, 1:1000) and pTyr1068-EGFR (No.3777, 1:1000) were purchased from Cell Signaling Technologies. Anti-GST (10000-0-AP, 1:4000), anti-Flag (66008-3-lg, 1:5000), anti-Myc (16286-1-AP, 1:2000), anti-UCH-L1 (66230-1-lg, 1:2000) and anti-β-actin (60008-1-lg, 1:5000) were purchased from Proteintech. Anti-eEF2K (ab45168, 1:1000) was purchased from Abcam. Anti-pThr678-EGFR (orb14895, 1:1000) was purchased from Biorbyt. Normal IgG/Peroxidase-conjugated AffiniPure Goat Anti-Rabbit/Mouse IgG (H+L) was purchased from Jackson Immuno Research.
9
Ubiquitin-mediated Protein Regulation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
pCDNA3 and pCDEF3 vectors obtained from Invitrogen and Addgene, respectively. Ubiqutin-His, Ubiqutin-HA, pIP-Usp7-Myc and pIP-Usp7 (C223S)-Myc plasmids were gifts from Dr. Bin Li. HA-PRK-Usp7 plasmid was a gift from Dr. Xingzhi Xu. pCDNA3-OGT-H508A plasmid was a gift from Dr. Jiemin Wong. The antibodies used are listed below: anti-MLL5 (AP14173a, Abgent), anti-FLAG M2 (F1804, Sigma), anti-HA antibody (3724, CST), anti-V5 (sc-271944, Santa Cruz), anti-Myc (16286-1-AP, Proteintech), anti-beta-Actin (A5316, Sigma), anti-ubiquitin (sc-8017, Santa Cruz), anti-OGT (Ab96718, Abcam), anti-O-Lined N-Acetylglucosamine (RL2) (ab2739, Abcam), anti-USP7 (3277, CST), anti-p27 antibody (610241, BD), anti-p16 antibody (sc-55600, Santa Cruz), anti-p21 antibody (2947, CST), anti-p14 (2407, CST), anti-Cyclin E (4126, CST). MG132 was purchased from Selleckchem (S2619). Cycloheximide was purchased from Sigma (C7698).
10
Investigating KLF13-TBX5 Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
293T cells were transfected with wild-type KLF13 or variants and TBX5 plasmids using FuGene HD according to the manufacturer's guidelines. Protein extracts were incubated overnight with anti-myc antibody (1:100, rabbit polyclonal anti-myc, 16286-1-AP, Proteintech, USA) coupled with magnetic beads.
Bound proteins were revealed with anti-TBX5 (1:1000, rabbit polyclonal anti-TBX5, 42-6500, Invitrogen, USA) antibody by Western blotting.
Bound proteins were revealed with anti-TBX5 (1:1000, rabbit polyclonal anti-TBX5, 42-6500, Invitrogen, USA) antibody by Western blotting.
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!