Cell and supernatant samples were separated by SDS-PAGE on an 11% SDS gel at 27 mA for 1 hour. Proteins were then transferred onto nitrocellulose membranes at 16 V over night and blocked for 1 hour in 1% skimmed milk in 0.1% Tween/PBS. Membranes were blotted with primary antibodies for 1 hour at room temperature and washed in 0.1% Tween/PBS. Subsequently, membranes were blotted with secondary conjugated antibody for 1 hour at room temperature and washed again before being visualised on a LI-COR Quantitative Imager. The following antibodies were used according to the manufactures’ instructions: anti-HSP90 sc7947 (Santa Cruz), anti-PKM2 ab38237 (Abcam), anti-HK2 ab104830 (Abcam), anti-GAPDH ab8245 (Abcam), anti-caspase-3 9668S (Cell Signaling), 800CW goat anti-rabbit 926–32211 (LI-COR) and 680RD goat anti-mouse 926–68070 (LI-COR). HIV-1 Gag was detected with supernatant from the mouse hybridoma 183-H12-5C, which was a kind gift from Prof. M. Malim.
800cw goat anti rabbit 926 32211
Manufactured by LI COR
Sourced in Denmark
The 800CW goat anti-rabbit 926–32211 is a near-infrared fluorescent secondary antibody. It is designed to detect and quantify the presence of rabbit primary antibodies in biological samples using near-infrared imaging systems.
Automatically generated - may contain errors
Lab products found in correlation
680rd goat anti mouse 926 68070, by LI COR (2 mentions)
Anti gapdh ab8245, by Abcam (1 mentions)
Anti hsp90 sc 7947, by Santa Cruz Biotechnology (1 mentions)
Sc 7947, by Santa Cruz Biotechnology (1 mentions)
Anti hsp90, by Santa Cruz Biotechnology (1 mentions)
Anti gfpt1, by Abcam (1 mentions)
Anti kga, by Proteintech (1 mentions)
Anti gac, by Proteintech (1 mentions)
Goat anti mouse 680rd, by LI COR (1 mentions)
Dapi d1306, by Thermo Fisher Scientific (1 mentions)
Mcherry ab167453, by Abcam (1 mentions)
Mab3408, by Merck Group (1 mentions)
4 protocols using 800cw goat anti rabbit 926 32211
1
Western Blot Analysis of Cellular Proteins
2
Quantitative Western Blot Analysis
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Cell and supernatant samples were separated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) on 11% gel for p24Gag and 15% gels, according to the size of the target proteins, at 27 mA per gel. Proteins were transferred onto nitrocellulose membrane overnight at 16 V and blocked for 1 h with 1% fat-free milk in 0.1% Tween/PBS. Membranes were incubated with primary antibodies at room temperature for at least 1 h.
The following primary antibodies were used: anti-HIV-1 Gag antibody (mouse hybridoma 183-H12-5C supernatant—kind gift from Prof. M. Malim), anti-GFPT1 (Abcam AB125069), anti-KGA (Proteintech 20170-1-AP), anti-GAC (Proteintech 19958-1-AP), anti-PPAT (OriGene TA504769S) and anti-HSP90 (Santa Cruz sc-7947). After incubation with primary antibodies, membranes were washed and incubated with secondary antibodies 680RD goat anti-mouse 926-68070 and 800CW goat anti-rabbit 926-32211 (both LI-COR) at room temperature for 1 h. Membranes were then washed again before visualization with a quantitative LI-COR imager.
The following primary antibodies were used: anti-HIV-1 Gag antibody (mouse hybridoma 183-H12-5C supernatant—kind gift from Prof. M. Malim), anti-GFPT1 (Abcam AB125069), anti-KGA (Proteintech 20170-1-AP), anti-GAC (Proteintech 19958-1-AP), anti-PPAT (OriGene TA504769S) and anti-HSP90 (Santa Cruz sc-7947). After incubation with primary antibodies, membranes were washed and incubated with secondary antibodies 680RD goat anti-mouse 926-68070 and 800CW goat anti-rabbit 926-32211 (both LI-COR) at room temperature for 1 h. Membranes were then washed again before visualization with a quantitative LI-COR imager.
3
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.
4
Multimarker Immunoblotting and Imaging
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Primary antibodies: anti-K V 11.1, APC-062 (Alomone, Jerusalem, Israel, 1:500); anti-PKD1, H00005587-A01 (Abnova, Roskilde, Denmark, 1:500); anti-β-tubulin, MAB3408 (Millipore, Hellerup, Denmark, 1:4000).
Secondary antibodies: For western blot analyses; peroxidase conjugated affinipure F(ab)fragment donkey anti-rabbit and anti-mouse (Jackson ImmunoResearch, Suffolk, UK), donkey anti-mouse 680RD, 926-68072 (LI-COR Biosciences, Copenhagen, Denmark); goat anti-rabbit 800CW, 926-32211 (LI-COR Biosciences). For confocal imaging; Alexa Fluor®488-conjugated donkey anti-rabbit IgG (1:200), Alexa Fluor®568-conjugated donkey anti-mouse IgG (1:200). Alexa®Fluor 647 Phalloidin (1:200) was used to stain actin filaments and DAPI (1:300) was used to stain the nucleus. Secondary antibodies were purchased from Invitrogen (Glostrup, Denmark).
Secondary antibodies: For western blot analyses; peroxidase conjugated affinipure F(ab)fragment donkey anti-rabbit and anti-mouse (Jackson ImmunoResearch, Suffolk, UK), donkey anti-mouse 680RD, 926-68072 (LI-COR Biosciences, Copenhagen, Denmark); goat anti-rabbit 800CW, 926-32211 (LI-COR Biosciences). For confocal imaging; Alexa Fluor®488-conjugated donkey anti-rabbit IgG (1:200), Alexa Fluor®568-conjugated donkey anti-mouse IgG (1:200). Alexa®Fluor 647 Phalloidin (1:200) was used to stain actin filaments and DAPI (1:300) was used to stain the nucleus. Secondary antibodies were purchased from Invitrogen (Glostrup, Denmark).
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