An antibody against GAPDH (MAB374) was from Millipore; Calnexin (ab22595) was from Abcam; Tubulin (DM1A) was from Sigma; CHMP2A (10477–1-AP) was from Proteintech; IST1 (51002–1-AP) was from Proteintech; CHMP7 (16424–1-AP) was from Proteintech; GFP (7.1/13.1) was from Roche; mCherry (ab167453) was from Abcam; HA.11 (16B12) was from (Biolegend); γH2AX (05–636) was from Sigma; 53BP1 (NB100-305) was from (Novus Biologicals). Anti-CDK1 substrate antibodies were from Cell Signaling Technology (9477S and 2325S). Anti-Histone H3 pS10 was from Cell Signaling Technology (9701S). Anti-LEM2 was from Sigma (HPA017340); anti-HSP90 (F8) was from Santa Cruz; anti-Emerin (10351–1-AP) was from Proteintech. Anti-LAP1 (21459–1-AP) was from Proteintech. Alexa conjugated secondary antibodies were from Invitrogen and HRP-conjugated secondary antibodies were from Millipore. IRDye 800 CW (925–32210) and IRDye 680 RD (925–68071) were from LI-COR Biosciences. Anti-peptide antibodies against CHMP7 pSer3 (3891 and 3892) were generated by immunisation of rabbits with KLH-conjugated peptides (MWpSPEREAEAPAGGC) by GenScript Biotech (Netherlands) B.V.
Mcherry ab167453
Manufactured by Abcam
Sourced in United Kingdom
MCherry (ab167453) is a fluorescent protein that emits light in the red-orange spectrum. It is commonly used as a reporter or tag in various biological applications to study protein localization, trafficking, and interactions.
Automatically generated - may contain errors
Lab products found in correlation
Rpb1 ntd d8l4y, by Cell Signaling Technology (2 mentions)
Anti histone h3 ps10, by Cell Signaling Technology (1 mentions)
γh2ax, by Merck Group (1 mentions)
Anti hsp90 f 8, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated secondary antibody, by Merck Group (1 mentions)
Anti emerin, by Proteintech (1 mentions)
Gfp 7.1 13 1, by Roche (1 mentions)
53bp1 nb100 305, by Novus Biologicals (1 mentions)
Klh conjugated peptides, by GenScript (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Irdye 680rd, by LI COR (1 mentions)
Alexa conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Calnexin, by Abcam (1 mentions)
800cw goat anti rabbit 926 32211, by LI COR (1 mentions)
Dapi d1306, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse 680rd, by LI COR (1 mentions)
T9284, by Merck Group (1 mentions)
Gfp a6455, by Thermo Fisher Scientific (1 mentions)
P0930, by Merck Group (1 mentions)
Nanog rcab0002p f, by Cosmo Bio (1 mentions)
7 protocols using mcherry ab167453
1
Antibody Validation and Characterization
2
Antibody Validation for Protein Analysis
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Affinity-purified rabbit antibodies against AnkB and GFP were generated in our laboratory and have been previously described (30 (link), 31 (link)). Other antibodies included mouse anti-alpha 1 sodium potassium ATPase (H-3, sc-48345), mouse anti-GFP (B-2, sc-9996), mouse anti-BCKDE1A (H-5, sc-271538), mouse anti-CD98 (E-5, sc-376815), mouse anti-FASN (G-11, sc-48357), and mouse anti-LAT1 (D-10, sc-374232) from Santa Cruz Biotechnology. Rabbit antibodies against mCherry (ab167453) and BCKDHA (phospho S293, ab200577) were purchased from Abcam. We also used rabbit anti-α-tubulin (11224-1-AP), rabbit anti Fabp4 (12802-1-AP), and mouse anti-GAPDH (2D4, 60004-I-AP) from Proteintech; rabbit anti-ASCT2 (ANT-082) from Alomone; and rabbit antibodies against GAPDH (D16H11 XP) and ACL (4332) from Cell Signaling Technologies. Secondary antibodies used for fluorescence imaging were purchased from Life Technologies and included donkey anti-rabbit IgG conjugated to Alexa Fluor 568 (#A10042) and donkey anti-mouse IgG conjugated to Alexa Fluor 568 (#A10037). Fluorescent signals in western blot analysis were detected using goat anti-rabbit 800CW (926–32211) and goat anti-mouse 680RD (926–68070) from LiCOR. Lipid droplets and nuclei were visualized by BODIPY™ 493/503 (D3922) and DAPI (D1306) staining, respectively, purchased from Life Technologies.
3
Multimodal Imaging of Proteins and Nucleic Acids
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Slides were blocked in 1%BSA/PBS/Triton X-100 0.1% for 30 min at 37°C before overnight incubation with the primary antibody at 4°C (Rpb1 NTD (D8L4Y) #14958, Cell Signaling Technology, 1 in 1000; mCherry [ab167453], abcam, 1 in 500). The following day, slides were washed in PBS before incubation with an appropriate secondary antibody (1 in 1000 Alexa Fluor) for 1 hr at 37°C. After further PBS washes and DAPI staining, slides were mounted with Vectashield.
For immuno-FISH (DNA), the IF signal was fixed via incubation with 4% PFA for 30 min. Following thorough PBS washes, the DNA FISH protocol was then followed as above.
For immuno-FISH (RNA), the antibodies were added at the same concentration as described above to the hybridization mix (primary antibody) and ×2 SSC/10% DF washes (secondary antibody).
For immuno-FISH (DNA), the IF signal was fixed via incubation with 4% PFA for 30 min. Following thorough PBS washes, the DNA FISH protocol was then followed as above.
For immuno-FISH (RNA), the antibodies were added at the same concentration as described above to the hybridization mix (primary antibody) and ×2 SSC/10% DF washes (secondary antibody).
4
Blastocyst Immunostaining for Lineage Markers
5
Western Blot Protein Detection Protocol
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For the detection of protein expression in total cell lysates, cells were lysed in Novex Tris-Glycine SDS sample buffer containing NuPAGE sample reducing agent (Invitrogen). Lysates were electrophoresed through Novex Tris-Glycine gels or NuPAGE Bis-Tris gels, transferred to nitrocellulose membranes, and subjected to immunoblot analysis. The blocking of membranes and subsequent antibody incubations were performed using Odyssey blocking buffer (LI-COR Biosciences) according to the manufacturer’s instructions. Primary antibodies against β-Tubulin (926–42211, LI-COR Biosciences), β-actin (926–42210, LI-COR Biosciences), FLAG (F1804, Millipore Sigma, Burlington, MA), mCherry (ab167453, Abcam, Cambridge, United Kingdom), GAPDH (ab8245, Abcam), and human SOD1 (ADI-SOD1–100, Enzo) were purchased from commercial sources. The IRDye 800CW-conjugated and IRDye 680-conjugated secondary antibodies were obtained from LI-COR Biosciences. Immunoblot signals were visualized by the Odyssey CLx infrared imaging system and quantified by ODYSSEY application software (LI-COR Biosciences).
6
B16 Melanoma Protein Profiling
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B16 melanoma cells were washed with ice-cold Phosphate buffer saline (PBS) and lysed with NP40 lysis buffer supplemented with protease inhibitor cocktail. Cells were incubated with NP40 for 30 minutes on ice. The soluble fractions of cell lysates were isolated by centrifugation at 13,000 rpm in a refrigerated microcentrifuge for 30 minutes. The protein concentration in the soluble fraction was quantified using the bicinchoninic acid (BCA) protein estimation kit.
Known concentrations of bovine serum albumin (BSA) was used to plot the standard curve.
30-50 μg of the protein was boiled in SDS dye and separated on 10% SDS PAGE gel.
Tyrosinase antibody is synthesized from Genescript. DCT (ab74073), PMEL17 (ab137078), MITF (ab12039), FASN (ab22759) and mCHERRY (ab167453) antibodies are procured from Abcam, CDK2 (MA1-81135) is obtained from Thermo Scientific while Srebf1 (04-469) is ordered from Millipore. HRP-conjugated Actin (ab8227) and Tubulin (ab6046) are used as a loading control. Horseradish peroxidase-conjugate Anti-Mouse (NA931) and Anti-Rabbit (NA934) antibodies are obtained from GE healthcare. For Western blot standard enhanced chemiluminescence reagents (WBLUF0100) were used from Millipore. ImageJ software was used for quantification.
Known concentrations of bovine serum albumin (BSA) was used to plot the standard curve.
30-50 μg of the protein was boiled in SDS dye and separated on 10% SDS PAGE gel.
Tyrosinase antibody is synthesized from Genescript. DCT (ab74073), PMEL17 (ab137078), MITF (ab12039), FASN (ab22759) and mCHERRY (ab167453) antibodies are procured from Abcam, CDK2 (MA1-81135) is obtained from Thermo Scientific while Srebf1 (04-469) is ordered from Millipore. HRP-conjugated Actin (ab8227) and Tubulin (ab6046) are used as a loading control. Horseradish peroxidase-conjugate Anti-Mouse (NA931) and Anti-Rabbit (NA934) antibodies are obtained from GE healthcare. For Western blot standard enhanced chemiluminescence reagents (WBLUF0100) were used from Millipore. ImageJ software was used for quantification.
7
Quantitative Immunofluorescence and FISH
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Slides were blocked in 1%BSA/PBS/Triton X-100 0.1% for 30 min at 37°C before overnight incubation with the primary antibody at 4°C (Rpb1 NTD (D8L4Y) #14958, Cell Signalling Technology, 1 in 1000; mCherry (ab167453), abcam, 1 in 500). The following day, slides were washed in PBS before incubation with an appropriate secondary antibody (1:1000 AlexaFluor)
for one hour at 37°C. After further PBS washes and DAPI staining, slides were mounted with Vectashield.
For immuno-FISH (DNA), the IF signal was fixed via incubation with 4% PFA for 30 minutes.
Following thorough PBS washes, the DNA FISH protocol was then followed as above.
For immuno-FISH (RNA), the antibodies were added at the same concentration as described above to the hybridization mix (primary antibody) and 2xSSC/10% DF washes (secondary antibody).
for one hour at 37°C. After further PBS washes and DAPI staining, slides were mounted with Vectashield.
For immuno-FISH (DNA), the IF signal was fixed via incubation with 4% PFA for 30 minutes.
Following thorough PBS washes, the DNA FISH protocol was then followed as above.
For immuno-FISH (RNA), the antibodies were added at the same concentration as described above to the hybridization mix (primary antibody) and 2xSSC/10% DF washes (secondary antibody).
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