Proteins extracted by RIPA lysis buffer and the samples from immunoprecipitation were separated by 12% SDS-PAGE and electro-blotted onto the PVDF membrane (Millipore, United States). Membranes were then blocked with 1% BSA for one hour and then incubated with the following primary antibodies overnight at 4°C: anti-acetyllysine antibody (1:5000 (V/V), PTM-101, PTM Biolab, China), CNPase rabbit monoclonal antibody (1:1000 (V/V), 5664, Cell Signal Technology), NEFL rabbit monoclonal antibody (1:500, 12998-I-AP, Proteintech, United States), and Gap 43 rabbit polyclonal antibody [1:1,000 (V/V), 16971-I-AP, Proteintech, United States]. Next, the membranes were washed three times with PBST. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibody [1:1,000 (V/V), Horse anti-mouse (7076S), Goat anti-rabbit (7074S), Cell Signal Technology], and for one hour at room temperature (RT). After washing, ECL (P0018FM, Beyotime Biotechnology, China) was added to visualize the blotted bands.
Horse anti mouse 7076
Manufactured by Cell Signaling Technology
Sourced in United States
Horse anti-mouse (7076S) is a secondary antibody product from Cell Signaling Technology. It is designed to detect mouse primary antibodies in various immunoassay applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit 7074, by Cell Signaling Technology (3 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Minimal essential amino acids, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
X ray film, by Fujifilm (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant, by Merck Group (1 mentions)
Mouse anti flag antibody f3165, by Merck Group (1 mentions)
Scrambled sirna, by Santa Cruz Biotechnology (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
5 protocols using horse anti mouse 7076
1
Western Blot Analysis of Neuronal Proteins
2
Characterization of Kv1.5 Channel Regulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Scrambled siRNA, primary antibodies against Vps24 (C6, sc-271501), N-terminal specific mouse anti-Kv1.5 antibody (A3, sc-377110), rabbit anti-Kv1.5 antibody (H-120, sc-25681), goat anti-mouse (sc-2005), and goat anti-rabbit (sc-2004) IgG-HRP secondary antibodies were purchased from Santa Cruz Biotechnology Inc. The rabbit C-terminal-specific anti-Kv1.5 antibody (APC-004) was purchased from Alomone Labs. The horse anti-mouse (7076S) and goat anti-rabbit (7074S) HRP-linked secondary antibodies were purchased from Cell Signaling Technology. MEM, FBS, trypsin, sodium pyruvate, minimal essential amino acids, hVps24 siRNA (ID: SASI_Hs01_00160243), Lipofectamine 2000, Opti-MEM, Hank's Balanced Salt Solution were purchased from Invitrogen. G418, PMSF, protease inhibitor cocktail, β-mercaptoethanol, Triton X-100, BSA, Prolong Gold Antifade mountant, monoclonal mouse anti-actin antibody (A4700), mouse anti-FLAG antibody (F3165), and all chemicals/electrolytes used were obtained from Sigma-Aldrich. The BLUeye Prestained Protein Ladder (GeneDirex) was purchased from FroggaBio. X-ray films were from Fujifilm. Paraformaldehyde was obtained from Alfa Aesar.
3
Pharmacological Modulation of hERG Channels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bisindolylmaleimide I (BIM-1, ab144264) was purchased from Abcam (Cambridge, MA). Gö 6983 (CAS no. 133053-19-7) and Sotrastaurin (STN, also known as AEB071, CAS no. 425637-18-9) were purchased from Cayman Chemical Company (Ann Arbor, MI). Goat anti-hERG (C20, sc-15968, C-terminal), mouse anti-hERG (F-12, sc-377388, N-terminal), and goat anti-integrin b1 (sc-6622) primary antibodies and mouse anti-goat IgG-HRP secondary antibody (sc-2354) were purchased from Santa Cruz Biotechnology (Dallas, TX). Mouse anti-hERG (DT-331, S5-Pore linker) antibody was purchased from D.I.V.A.L. (Toscana S.R.L., Florence, Italy). Mouse anti-actin primary antibody (A4700) was purchased from Sigma-Aldrich. Rabbit antiphosphorylated Nedd4-2 (p-Nedd4-2, Ser-448, 8063S) primary antibody, horse anti-mouse (7076S), and goat anti-rabbit (7074S) secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA). The PKC sampler kit containing isoenzyme specific antibodies (611421) was obtained from BD Biosciences (Franklin Lakes, NJ). Electrolytes and chemicals for patch-clamp recordings were purchased from Sigma-Aldrich.
4
Quantifying Muscle Protein Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The gastrocnemius muscle samples were homogenised in buffer containing 20 mM Tris, 1 mM DTT, 2 mM ATP, and 5 mM MgCl2, centrifuged at 12,000 g, and total protein content was assessed using the Lowry method [32 (link)]. Muscle samples (60 µg of protein) were analysed using SDS-polyacrylamide gel electrophoresis (10% or 12%) and transferred to a 0.45-µm pore size nitrocellulose membrane, which was blocked with skim milk (5%) for 1 h. Proteins were probed with primary antibodies GAPDH (SC47724) (Santa Cruz Biotechnology, Santa Cruz, CA, USA); 20S (PW8195), 19S (PW9265), and 11S (PW8185) (Enzo Life Sciences, Farmingdale, NY, USA); PI3K (4292), phosphor-PI3K (4228), mTOR (2972), phospho-mTOR (2971), p70S6K (9202), phospho-p70 S6KThr421/Ser424 (9204), 4E-BP1 (9452), phospho-4E-BP1Thr70 (9455), and eIF4G (2498) (Cell Signalling, Danvers, MA USA); and secondary antibodies goat anti-rabbit (7074) and horse anti-mouse (7076) (Cell Signalling). The Western blot band images were captured using the Alliance 2.7 (UVITEC, Cambridge, UK) and quantified using UVIband-1D (UVITEC), and protein expression was normalised using GAPDH as a loading control.
5
Western Blotting of α-SMA and Vinculin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LX-2 cells were harvested after 24 h incubation time at either 1 or 10 % FBS at ~85 % confluency using trypsin-EDTA. Cells were lysed in CST lysis buffer (Cell Signaling Technologies, Danvers, MA, USA) supplemented with protease inhibitor cocktail (Sigma Aldrich). SDS-PAGE (NuPage, Thermo Fisher, Waltham, MA, USA) was used to resolve equal amounts of protein. Semi-dry blotting was performed onto nitrocellulose membranes (Amersham, GE Healthcare, Chicago, IL, USA). Blocking of membranes was achieved by 5% skim milk in TBS-T for 30 min at RT. Primary antibody (α-SMA, Invitrogen 1A4; vinculin, Invitrogen 7F9) incubation was performed over night at 4 °C and secondary HRP antibody (horse anti-mouse 7076, Cell Signaling Technologies) incubation was performed at RT for 1 h. Chemiluminescent detection agent Pierce ECL PLUS (Thermo Fisher) was used as substrate and detection was done at a ChemiDoc MP (BioRad, Hercules, CA, USA). Image analysis was carried out with Image Lab (BioRad).
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!