Protein lysates were extracted using RIPA lysis buffer (Pierce, Thermo Fisher Scientific). Protein estimation was conducted using a BCA protein assay kit (Pierce). Protein lysates were denatured and subjected to SDS-PAGE electrophoresis. Proteins were then transferred onto a nitrocellulose membrane and blocked with 5% non-fat milk, followed by the incubation with primary antibody at 4 °C overnight. Membranes were then rinsed and incubated with a corresponding secondary antibody (1:5000, Invitrogen) at room temperature for 1 h. Signal was visualized using Pierce ECL plus Western blotting substrate (Pierce). The following primary antibodies were used in this study: anti-POU1F1 (1:3000; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-HA tag (1:2000; Invitrogen); anti-V5 tag (1:2000; Invitrogen); anti-CD163 (1:5000; Abcam); anti-CD206 (1:5000; Sigma–Aldrich, St Louis, MO, USA), anti-CD11b (Novus Biologicals, Centennial, CO, USA); anti-VEGFA (1:5000; Santa Cruz); anti-p-CXCR4 (1:1000; Abcam, Cambridge, UK); anti-CXCR4 (1:1000; Abcam); anti-p-Akt (1:1000; Cell signaling technology, Beverly, MA, USA); anti-Akt (1:1000; Cell signaling technology); anti-p-VEGFR2 (1:1000; Cell signaling technology); anti-VEGFR2 (1:1000; Cell signaling technology); anti-GAPDH (1:3000; Santa Cruz).
Anti ha tag
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-HA tag is a laboratory tool used for the detection and purification of proteins that have been tagged with the HA (Hemagglutinin) epitope. The HA tag is a short amino acid sequence that can be added to a protein of interest, allowing it to be identified and isolated using specific antibodies against the HA tag.
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Lab products found in correlation
Supersignal west pico chemiluminescent substrate, by GE Healthcare (3 mentions)
Nupage sds page, by Thermo Fisher Scientific (3 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (3 mentions)
Anti flag tag, by Merck Group (3 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Anti pgk1, by Thermo Fisher Scientific (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Anti flag m2 tag antibodies, by Merck Group (2 mentions)
Anti gapdh antibody, by Merck Group (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Taqman probe, by Thermo Fisher Scientific (2 mentions)
Anti v5 tag, by Thermo Fisher Scientific (1 mentions)
Anti vegf a, by Santa Cruz Biotechnology (1 mentions)
Anti p vegfr2, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti p akt, by Cell Signaling Technology (1 mentions)
Anti cd11b, by Novus Biologicals (1 mentions)
15 protocols using anti ha tag
1
Western Blot Analysis of Protein Expression
2
Apoptosis Evaluation in p75NTR KO Neurons
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For assessing apoptosis, p75NTR KO neurons transfected with the different plasmids (see above) were cultured for 2 days, fixed with solution containing 4% paraformaldehyde and 4% sucrose. The fixed cells were labeled with cleaved caspase 3 (Cell signal, 9761, 1:400), anti HA-tag (Invitrogen, 71–5500, 1:250), and DAPI. For each experiment, neurons were culture in duplicates and at least 15 images were taken per coverslip.
3
Analyzing Mouse Glomerular Proteins
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The primary antibodies used in this study included anti‐WT1 (polyclonal: c‐19 and monoclonal: F6, Santa Cruz Biotechnology), anti‐NF‐kB p50 (Santa Cruz Biotechnology), anti‐GST (cell signaling), anti‐HA‐Tag (cell signaling), and anti‐V5 (Invitrogen). Control IgGs were purchased from Alpha Diagnostic International (San Antonio, Texas, USA).
For the preparation of mouse glomerular extract, fractions of kidney cortex were enriched in glomeruli by successive sieving through 105‐, 75‐, and 40‐μm cell strainers, as previously reported.23 Glomerular protein extracts were prepared in lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, 1 mM protease inhibitors, 1 mM NaF and 1 mM sodium orthovanadate). The protein lysates were resolved by SDS‐PAGE and analyzed by Western blot with the indicated antibodies.
For the preparation of mouse glomerular extract, fractions of kidney cortex were enriched in glomeruli by successive sieving through 105‐, 75‐, and 40‐μm cell strainers, as previously reported.
4
Antibody panel for cell signaling study
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Anti PI3KC2α (#22028‐1‐AP, Proteintech), anti GFP (gift from Emilia Turco, University of Turin, Italy), anti α‐tubulin (#2125, Cell Signaling), anti GAPDH (sc‐47724, Santa Cruz Biotechnology), anti Myc‐tag (#2276, Cell Signaling), anti FAK (#71 433, Cell Signaling), anti p‐FAK (tyr397) (#8556, Cell Signaling), anti p‐FAK (tyr925) (#3284, Cell Signaling), anti Paxillin (#2542, Cell Signaling), anti p‐Paxillin (tyr118) (#69 363, Cell Signaling), anti HA‐tag (# 26 183, Thermofisher), anti R‐RAS (#8446, Cell Signaling), anti RASA3 (#PA5‐30445,Invitrogen), anti Rap1A/Rap1B (#4938, Cell Signaling), anti RAS (#3339, Cell Signaling), and anti Vinculin (#V9131, Sigma).
5
Immunofluorescence Imaging of Transfected Cells
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Transfected cells were harvested 24 h post-transfection. Cells were washed once with PBS and fixed with 500 μL of 4% paraformaldehyde/2.5% sucrose solution for 10 min at RT followed by three washing steps in PBS. Fixed cells were permeabilized by 10 min incubation in 500 μL of 0.1% Triton X-100 in PBS and washed two times with PBS. Blocking was performed by incubation in 500 μL PBS containing 2% BSA (Sigma Aldrich) for 30 min. Afterward, primary antibodies were used at the following dilutions: anti-HA tag (Thermo Fisher Scientific; mouse, 1:100, 26183-D488) and anti-calreticulin antibody (Abcam; rabbit, 1:500, ab92516) diluted in PBS containing 1% BSA. After 60 min incubation and washing three times with PBS, cells were incubated with secondary antibodies using Alexa Fluor 568 (Thermo Fisher Scientific; donkey anti-rabbit, 1:1,000, dA10042) diluted in PBS containing 3% BSA for 60 min in the dark. Nuclei were stained with DAPI by using the mounting medium Roti-Mount FluorCare DAPI (Roth) on glass slides. Samples were examined using the Axio observer microscope (Zeiss). Images were edited and merged by using the ZEN 2.3 software.
6
Western Blot Analysis of Apoptosis Markers
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Cells were collected from each group by washing three times with PBS. The total protein was extracted by adding ice-cold RIPA lysis buffer in the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). The protein was transferred to the nitrocellulose membrane after 12% SDS-PAGE electrophoresis. Then the membranes were blocked with tris-buffered saline (TBS) and Tween-20 (Thermo Fisher Scientific, Waltham, MA, USA) containing 5% fat-free-milk for 1 hour at room temperature. After the primary antibody was added, it was incubated overnight at 4°C. Primary antibodies included: anti-Bcl-2, anti-Bax, anti-Cleaved Caspase 9 (Santa Cruz, Dallas, TX, USA), anti-ERK1, anti-Phospho-ERK anti-Collagen II, anti-β-actin, anti-GAPDH (Abcam, Cambridge, MA, USA), and anti-HA-Tag (Thermo Fisher Scientific, Waltham, MA, USA). After the membranes were washed the next day, the secondary antibody (Bioworld) was incubated for 1 hour at room temperature and PBST washed again. ECL chemiluminescent substrates were added to it and X-ray was used to expose. The relative expression of the target protein was evaluated by comparing the gray value ratio of target protein content to the β-actin content (target protein/β-actin) using the Quantity One 4.62 image analysis software.
7
Comprehensive Protein Analysis Protocol
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Western blotting was performed as previously described [37 (link)]. In addition, cytoplasmic and nuclear extracts were separated and prepared using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions and a previous study [36 (link)]. Primary antibodies (anti-E-cadherin [Cat# ab15148, 1:500], anti-N-cadherin [Cat# ab18203, 1:1000], anti-ZEB1 [Cat# ab124512, 1:1000], anti-ZEB2 [Cat# ab138222, 1:1000], anti-peroxiredoxin 1/PAG [Cat# ab109498, 1:10,000] and anti-GAPDH [Cat# ab181602, 1:1000] [Abcam, USA]; anti-Vimentin [Cat# D21H3, 1:1000, CST, USA]; anti-H3 [Cat# AH433, 1:1000, Beyotime, China]; and anti-HA Tag [Cat# 26183, 1:10000, Thermo Fisher Scientific]) were used, as well as anti-mouse and anti-rabbit secondary antibodies (IR Dye-labeled secondary antibodies [1:10000, Sigma, USA] and HRP-labeled secondary antibodies [1:10000, CST]). The signals were visualized using an Odyssey Infrared Imaging System (LI-COR Biosciences, USA) or with ECLUltra (New Cell and Molecular Biotech, Suzhou, China).
8
Western Blot Analysis of Tagged Proteins
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Protein extracts were made as described and 20 μg of protein was separated by 4%–12% NuPAGE SDS-PAGE (Thermo) [48 (link)]. Proteins were detected using the following antibodies; anti-HA tag (Thermo #26183), Anti-FLAG tag (Sigma, #F1365), anti-PGK1 (Thermo # PA5-28612), anti-Ydj1 (StressMarq #SMC-166D). Blots were imaged on a ChemiDoc MP imaging system (Bio-Rad). After treatment with SuperSignal West Pico Chemiluminescent Substrate (GE). Blots were stripped and re-probed with the relevant antibodies using Restore Western Blot Stripping Buffer (Thermo).
9
Comprehensive Cell Signaling Assay Protocol
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RQ1 RNase‐free DNaseI (Promega, #M6101), SuperScript III First strand‐synthesis system (Thermo Fisher Scientific, #18080051), RNase A (Merck, #10109242001), anti‐EGFP (Abcam, UK, #ab290), anti‐SRSF3 (Merck, #WH0006428M8), anti‐CDKN1A (Cell Signalling Technologies, #2947), anti‐GAPDH (Cell Signalling Technologies, #2118S), anti‐TP53 (Cell Signalling Technologies, #2524), anti‐HA tag (ThermoFisher Scientific, #MA5‐25644), TaqMan® MicroRNA Reverse Transcription Kit (ThermoFisher Scientific, #4366596), SensiFAST Probe Hi‐ROX Kit (Bioline, #BIO‐82005), Pierce Firefly Luciferase Glow Assay Kit (ThermoFisher Scientific, #16176), Dynabeads Protein G beads (Thermo Fisher Scientific, #10009D), TRI Reagent (Sigma‐Aldrich, #T9424), Luminaris HiGreen qPCR Master Mix‐low ROX (ThermoFisher Scientific, #K0974), predesigned TaqMan probes (ThermoFisher Scientific, #4427975). NuPAGE 4–12% gradient Bis‐Tris gels (ThermoFisher Scientific, #NP0322BOX), ECL Western Blotting Detection Reagents (MERCK, #GERPN2209), Silencer Select predesigned siRNA (ThermoFisher Scientific, #4392420, SRSF3 assay ID s12732 and Negative Control No. 1), 2‐methylnicotinic acid imidazolide (MERCK, #913839), eBioscience™ Cell Proliferation Dye eFluor™ 670 (ThermoFisher Scientific, #65‐0840‐85) and cOmplete™ Mini EDTA‐free (Merck, #11836170001).
10
Protein Detection and Antibody Probing
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First, 20 µg of protein was separated by 4–12% NuPAGE SDS-PAGE (Thermo Fisher Scientific). Proteins were detected using the following antibodies; anti-HA tag (Thermo Fisher Scientific), Anti-FLAG tag (Sigma-Aldrich, St. Louis, MO, USA, USA #F1365), anti-PGK (Thermo Fisher Scientific, #MA5-15738), anti-Ydj1 (Stressmarq Biosciences Inc., Victoria, BC, Canada, #SMC-166D), anti-HDJ2 (Thermo Fisher Scientific, #MA512748). Blots were imaged on a ChemiDoc MP imaging system (Bio-Rad, Hercules, CA, USA). After treatment with Super Signal West Pico Chemiluminescent Substrate (GE Healthcare, Piscataway, NJ, USA). Blots were stripped and reprobed with the relevant antibodies using Restore Western Blot Stripping Buffer (Thermo Fisher Scientific).
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