Whole-cell lysates and immunoblotting were performed as described elsewhere (19 (link), 20 (link)). Blots were probed with the following antibodies obtained from Santa Cruz or CST (Danvers, MA, USA): phospho-ERK: Cat# sc-7383, RRID AB_627545, 1:1000; ERK: Cat# sc-514302, RRID : AB_2571739, 1:1000; SHP2: Cat# 3397, RRID: AB_2174959, 1:1000; PDGFRβ: Cat# sc-374573, RRID: AB_10990921, 1:100; pan-RAS: Cat# sc-166691, RRID: AB_2154229, 1: 200; GAPDH: Cat# sc-47724, RRID: AB_627678, 1:2000; anti-rabbit: Cat# 926-32211, RRID: AB_621843; Cat# 926-926-68071, RRID: AB_10956166; anti-mouse: Cat# 926-32210, RRID: AB_621842, Cat# 926-680707, RRID: AB_10956588; all 1:10,000, LiCOR (Bad Homburg, Germany). Primary antibodies were diluted in Intercept/TBS blocking solution (LiCOR) supplemented with 0.2% Tween-20, secondary antibodies were diluted in TBS supplemented with 0.1% Tween-20. Total protein staining was performed using Revert 700 Total Protein Stain Solution according to the manufacturer’s protocol (LiCOR). Densitometry was performed using Empiria Studio 1.2 (LiCOR).
Intercept tbs blocking solution
Manufactured by LI COR
Intercept/TBS blocking solution is a laboratory reagent used to prevent non-specific binding in Western blot and other immunoassay techniques. It is designed to block unoccupied binding sites on the membrane or solid support, reducing background signal and improving the specificity of target detection.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Gapdh, by LI COR (2 mentions)
Revert 700 total protein stain solution, by LI COR (1 mentions)
Empiria studio 1, by LI COR (1 mentions)
Anti mouse, by LI COR (1 mentions)
Anti rabbit, by LI COR (1 mentions)
Glut1, by Santa Cruz Biotechnology (1 mentions)
Hif 1α, by R&D Systems (1 mentions)
Beta actin, by BioLegend (1 mentions)
Foxp1, by Cell Signaling Technology (1 mentions)
α tubulin, by Synaptic Systems (1 mentions)
Vegfa, by Proteintech (1 mentions)
Plasma membrane protein extraction kit, by Abcam (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
3 protocols using intercept tbs blocking solution
1
Immunoblotting Assay for Signaling Proteins
2
Protein Extraction and Western Blot Analysis of Mouse Embryonic Stem Cells
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Whole-cell extracts from mESCs cultured in feeder-free 2i media were prepared in a modified RIPA lysis buffer: 50 mM Tris (pH 7.4), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, and 1x SigmaFast protease inhibitor cocktail. Spheroids were lysed in RIPA buffer and protease inhibitors. Protein concentration was determined with a BCA kit (Thermo Fisher Scientific) and normalized to 1.0–2.0 μg/μl. 20–25 μl of protein was mixed with sample buffer and 50–100 mM DTT. Protein was transferred to a PVDF membrane, blocked with either 1% BSA or Intercept (TBS) Blocking Solution (LI-COR), and blotted with primary antibodies at 4 °C overnight. Primary antibodies used: Foxp1 (1:1000, D35D10, #4402, Cell Signaling) (Glut1 (2 μg/ml, #sc-377228, Santa Cruz Biotechnology), HIF-1α (1 μg/ml, #AF1935, R&D Systems), Ldha (1:1000, #2012 Cell Signaling), Vegfa (1:500, #19003, Proteintech), Beta-Actin (1 μg/ml, 664802, Biolegend), Gapdh (1:1000, #926-422116, LI-COR), α-Tubulin (Synaptic Systems, 1:2000, #302 204). Western blots were imaged on an Odyssey DLx Imager system.
3
Immunoblotting Protocol for Membrane Fractions
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Whole cell lysates and immunoblotting were performed as previously described (17 (link),23 (link),24 (link)). For membrane fractionation, the Plasma Membrane Protein Extraction kit (Abcam) was used. A total of 20 µg of protein was loaded onto the respective membranes and blots were probed with antibodies obtained from Cell Signaling Technology, Inc. [(p-ERK: cat. no. 9102; RRID: AB_330744; 1:2,000), (p-NFκB: cat. no. 93H1; RRID: AB_10827881; 1:1,000) NFκB: cat. no. D14E12; RRID: AB_10859369; 1:1,000) p-p38: cat. no. 9211, RRID: AB_331641; 1:500)], Santa Cruz Biotechnology, Inc. [(ERK: cat. no. sc-514302; RRID: AB_2571739; 1:750; FN1: cat. no. sc-8422; RRID: AB_627598; 1:200), GAPDH: cat. no. sc-47724; RRID: AB_627678; 1:1,000), (p38: cat. no. sc-7972; RRID: AB_628079; 1:1,000)] or LI-COR Biosciences [(anti-mouse: cat. no. 926-32210; RRID: AB_621842; cat. no. 926-680707; RRID: AB_10956588), anti-rabbit: cat. no. 926-68071; RRID: AB_10956166; cat. no. 926-32211; RRID: AB_621843; all 1:10,000]. Primary antibodies were diluted in Intercept/TBS blocking solution (LI-COR Biosciences) supplemented with 0.2% Tween-20, secondary antibodies were diluted in TBS supplemented with 0.1% Tween-20.
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