Clean blot

Manufactured by Thermo Fisher Scientific

Clean-Blot is a laboratory equipment designed for efficient removal of unwanted proteins from Western blot membranes. It provides a simple and effective method to remove non-specific background signals, improving the clarity and specificity of Western blot analysis.

Automatically generated - may contain errors

Lab products found in correlation

Spelling variants of this product

Clean-Blot IP Detection Reagent (47 citations) Clean-Blot IP Detection Reagent (HRP) (15 citations) Clean-Blot IP Detection Kit (10 citations) Clean-Blot™ IP Detection Kit (HRP) (5 citations) HRP-conjugated Clean-blot IP detection reagent (2 citations) Clean-blot HRP (2 citations) Clean Blot IP Detector Reagent (2 citations)

6 protocols using clean blot

Immunoblotting and Immunofluorescence Antibodies

Check if the same lab product or an alternative is used in the 5 most similar protocols

The following antibodies were used for immunoblotting and immunoprecipitation: anti-IRE1β (Bertolotti et al., 2001 (link)), anti–phospho-IRE1β (gift from K. Kohno, Nara Institute of Science and Technology, Takayama, Ikoma, Nara, Japan), anti-IRE1α (20790; H-190; polyclonal; Santa Cruz Biotechnology, Inc.), anti-IRE1α (3294; 14C10), anti-NDP52 (9036; polyclonal), anti-NBR1 (9891; D2E6), anti-GAPDH (2118; 14C10), anti–β-actin (4970; 13E5; Cell Signaling Technology), antioptineurin (100000; polyclonal; Cayman), and anti-p62 (PM045; polyclonal; MBL). Immunofluorescence antibodies were anti-IRE1α (20790; H-190), antilysozyme (27958; C19), anti-MUC2 (15334; H-300), anti-IRE1β (Bertolotti et al., 2001 (link); Santa Cruz Biotechnology, Inc.), and anti-GRP78 (ab21685; polyclonal; Abcam). The following secondary antibodies were used: donkey anti–rabbit IgG (DyLight 488; ab96919; polyclonal), donkey anti–goat IgG (DyLight 488; ab96931; polyclonal), donkey anti–rabbit IgG (DyLight 650; ab96894; polyclonal; Abcam), and Clean-Blot (21230; Thermo Fisher Scientific). UEA-1 (RL-1062; Vector Laboratories) was used to stain secretory IECs.

Tschurtschenthaler M., Adolph T.E., Ashcroft J.W., Niederreiter L., Bharti R., Saveljeva S., Bhattacharyya J., Flak M.B., Shih D.Q., Fuhler G.M., Parkes M., Kohno K., Iwawaki T., Janneke van der Woude C., Harding H.P., Smith A.M., Peppelenbosch M.P., Targan S.R., Ron D., Rosenstiel P., Blumberg R.S, & Kaser A. (2017). Defective ATG16L1-mediated removal of IRE1α drives Crohn’s disease–like ileitis. The Journal of Experimental Medicine, 214(2), 401-422.

+ Open protocol

+ Expand

Co-Immunoprecipitation of PKA Signaling Complex

Check if the same lab product or an alternative is used in the 5 most similar protocols

Protein A/G PLUS-Agarose beads (Santa Cruz sc-2003) or Protein A/G Magnetic Beads (bimake.com B23202) were equilibrated in Tris buffer. Pre-clearing was achieved by incubation of agarose beads with 1 mg of nuclear protein and 5 μg rabbit IgG (Santa Cruz sc-2027) for 1 h at 4°C to remove proteins that nonspecifically bind the beads or IgG. Pre-cleared lysate was incubated with the immunoprecipitating antibody for 1 h at 4°C. The lysate-antibody mix was incubated with agarose beads overnight. After 4 washes with ice-cold DPBS, bound protein was eluted by boiling for 5 min in SDS sample buffer. Antibodies used for Co-IP: PKA IIα reg (C-20) (sc-908), PDE4D5 (gift from Dr. George Baillie’s Lab), AKAP95 (R-146) (sc-10766), Clean Blot (Thermo Fisher Scientific 21230).

Clister T., Greenwald E.C., Baillie G.S, & Zhang J. (2019). AKAP95 organizes a nuclear microdomain to control local cAMP for regulating nuclear PKA. Cell chemical biology, 26(6), 885-891.e4.

+ Open protocol

+ Expand

Immunoprecipitation and Western Blot Analysis of GTPase-Subunit Interactions

Check if the same lab product or an alternative is used in the 5 most similar protocols

HEK293T cells were transfected with plasmids encoding a FLAG-tagged subunit a isoform and a V5-tagged small GTPase, lysed in IP buffer (1% Triton X-100, 10% glycerol, 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride and protease inhibitor cocktails) and immunoprecipitated with an anti-FLAG antibody as described previously^{30 ,72 (link)}. Immunoprecipitates were analysed by Western blotting using Clean Blot (Thermo Scientific) as a secondary antibody. HRP-conjugated host-specific secondary antibodies (GE Healthcare) were used for Western blotting of macrophage and osteoclast lysates. Immune complexes were detected by chemiluminescence using an ECL prime detection kit (GE Healthcare) and an LAS-3000 imaging system (FUJIFILM).

Matsumoto N., Sekiya M., Tohyama K., Ishiyama-Matsuura E., Sun-Wada G.H., Wada Y., Futai M, & Nakanishi-Matsui M. (2018). Essential Role of the a3 Isoform of V-ATPase in Secretory Lysosome Trafficking via Rab7 Recruitment. Scientific Reports, 8, 6701.

+ Open protocol

+ Expand

Western Blotting Analysis for BCNT-C

Check if the same lab product or an alternative is used in the 5 most similar protocols

Western blotting analysis was carried out essentially as described previously [17 (link)] except for two reagents: the second antibody of Clean-Blot (Thermo Fisher Scientific) for guinea pig anti-BCNT-C antibody and the substrate of the nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) solution (Roche) or its tablets (Sigma) for development. For reprobing, the filter was treated with 2% SDS, 80 mM 2-mercaptoethanol in 62. 5 mM Tris/HCl (pH7.4) at 50°C for 30 min. After rinsing with H₂O, the filter was dipped twice in dimethylformamide for 10 s at room temperature, rinsed again with H₂O and subjected to blocking as usual. An anti-BCNT-C peptide antibody [17 (link)] and an anti-6× histidine monoclonal antibody (9C11, Wako) were used.

Iwashita S., Suzuki T., Yasuda T., Nakashima K., Sakamoto T., Kohno T., Takahashi I., Kobayashi T., Ohno-Iwashita Y., Imajoh-Ohmi S., Song S.Y, & Dohmae N. (2015). Mammalian Bcnt/Cfdp1, a potential epigenetic factor characterized by an acidic stretch in the disordered N-terminal and Ser250 phosphorylation in the conserved C-terminal regions. Bioscience Reports, 35(4), e00228.

+ Open protocol

+ Expand

Antibody sources and conjugates

Check if the same lab product or an alternative is used in the 5 most similar protocols

Information of antibodies can be found in Supplementary Table S2 online. Antibodies against FLAG, β-actin, and the B2 subunit of V-ATPase were purchased from Sigma-Aldrich. Antibodies to V5, Ccz1, CD68, and the A subunit of V-ATPase were obtained from Thermo Scientific, Santa Cruz, Hycult Biotech, and Abcam, respectively. Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgGs were from GE Healthcare. Clean-Blot, an HRP-conjugated antibody for post-immunoprecipitation western blot detection of native primary antibodies, was purchased from Thermo Scientific. Alexa-conjugated secondary antibodies were from Thermo Scientific.

Matsumoto N., Sekiya M., Sun-Wada G.H., Wada Y, & Nakanishi-Matsui M. (2022). The lysosomal V-ATPase a3 subunit is involved in localization of Mon1-Ccz1, the GEF for Rab7, to secretory lysosomes in osteoclasts. Scientific Reports, 12, 8455.

+ Open protocol

+ Expand

Immunoprecipitation and Western Blot Analysis of KMT2D and SGK Signaling

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors and separated using SDS-PAGE gradient gels (4%–12%). The KMT2D probed gels were separated using SDS-PAGE gradient 3%–8%. After transfer to a PVDF membrane for 2 hours at 70 V, the membranes were probed using Vinculin, Actin, SGK1, SGK3, pNDRG1 (T346) and phospho-RXRXX(S/T); all were from Cell Signaling Technology (CST). The rabbit KMT2D was generated by Eurogentec (third bleed) and affinity purified against Ac-GRARLKSTASSI C-NH2 while the rabbit pKMT2D (S1331) was affinity purified against Ac-GRARLKS(PO₃H₂)TASSIC-NH2 peptide.
Cell lysates for co-immunoprecipitation assays were made using NP-40 buffer and incubated overnight with EZview Anti-HA agarose beads. The immunocomplexes were then washed three times with NP40 and immunoblotted with the indicated antibodies. For endogenous co-immunoprecipitation of SGK1 and KMT2D, we employed the KMT2D antibody A300BL1185 (Bethyl) in 5 mg of JIMT1 lysate and SGK1 was recognized using the CST antibody with a secondary conformational specific antibody from Clean Blot (Thermo).

Toska E., Castel P., Chhangawala S., Arruabarrena-Aristorena A., Chan C., Hristidis V.C., Cocco E., Sallaku M., Xu G., Park J., Minuesa G., Shifman S.G., Socci N.D., Koche R., Leslie C.S., Scaltriti M, & Baselga J. (2019). PI3K Inhibition Activates SGK1 via a Feedback Loop to Promote Chromatin-Based Regulation of ER-Dependent Gene Expression. Cell reports, 27(1), 294-306.e5.

+ Open protocol

+ Expand

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?

Revolutionizing how scientists
search and build protocols!