Liposome flotation assays were performed as described previously (Chowdhury et al., 2018 (link)). Briefly, ATG2A, WIPI4, and WIPI1 proteins were mixed with 25 µM LUVs (5% PI3P, 49% DOPC, 25% DOPE, 20% DOPS, and 1% 1,1’-Dioctadecyl-3,3,3’,3’-tetramethylindodicarbocyanine perchlorate (DiD) (Marker Gene Technologies)) as indicated in Figure 5B and C in 150 µL of buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM TCEP, and 40% Nycodenz at the bottom of centrifugation tubes. A layer of 400 µL of 30% Nycodenz buffer was placed on the top of each sample, and a layer of 50 µL of 0% Nycodenz buffer was then placed on top. Tubes were centrifuged as described above. The top (150 µL), middle (300 µL), and bottom (150 µL) fractions were collected from the top and subjected to western blotting. ATG2A-TwinStrepII and His-WIPI proteins (10 × His-WIPI4 and 6 × His-WIPI1) were probed with rabbit anti-StrepII (GenScript) and mouse anti-His tag (Millipore) antibodies, respectively. Goat anti-mouse IRDye 800CW and goat anti-rabbit IRDye 680 secondary antibodies (LI-COR Biosciences) were used for infrared fluorescence detection of the bands on LI-COR Odyssey scanner (LI-COR Biosciences).
Mouse anti his tag
Manufactured by Merck Group
Sourced in Germany
The Mouse anti-His tag is a laboratory reagent used to detect and purify recombinant proteins containing a histidine (His) tag. The His tag is a commonly used affinity tag that allows for the rapid identification and purification of target proteins expressed in various expression systems. The Mouse anti-His tag antibody specifically binds to the His tag, enabling researchers to effectively isolate and study their proteins of interest.
Automatically generated - may contain errors
Lab products found in correlation
Aquavivid, by Thermo Fisher Scientific (2 mentions)
Anti sars cov 1 spike cr3022, by Merck Group (2 mentions)
Alexa fluor 647 conjugated goat anti human igg h l abs, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse igg h l abs, by Thermo Fisher Scientific (2 mentions)
Lsrii cytometer, by BD (2 mentions)
Odyssey scanner, by LI COR (1 mentions)
Western lightning plus ecl, by PerkinElmer (1 mentions)
Goat anti mouse hrp, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Prestained protein ladder, by Sinaclon (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Pcr master mix, by GoldBio (1 mentions)
1 kb dna ladder, by GoldBio (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Sinaclon (1 mentions)
Hrp conjugated anti mouse igg antibody, by Merck Group (1 mentions)
Ampicillin, by Merck Group (1 mentions)
Lb agar, by BD (1 mentions)
6 protocols using mouse anti his tag
1
Liposome Flotation Analysis of ATG2A, WIPI4, and WIPI1
2
Recombinant Trefoil Factor 1 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The rTFF1 produced by E. coli BL21(DE3) (pET-TFF1) and B. choshinensis (pNCMO2-TFF1) at various time expression intervals were analyzed by Tricine-SDS-PAGE [37 (link)]. The yield of rTFF1 was estimated by comparing the density of the rTFF1 bands to standards (serial dilutions of 103 mg/L purified rTFF1) on SDS-PAGE gels analyzed using Gel-Pro AnalyzerTM version 3.0 (Total-Integra Technology Co., Ltd, Taipei, Taiwan). The electrophoresis gel was then transferred into PVDF membranes (Millipore, Darmstadt, Germany). The mouse anti- His•tag (Millipore, Darmstadt, Germany) and goat anti-mouse HRP (Millipore, Darmstadt, Germany) were used to immunize the rTFF1. The presentations of rTFF1s were then stained with Western LightningTM Plus-ECL (Perkin Elmer, Inc., USA), analyzed by Fusion-Capt advance analysis software (Vilber Lourmat, France).
3
Quantifying SARS-CoV Spike Expression
4
Detecting Coronavirus Spike Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Spike expressors of human coronaviruses SARS-CoV-2, SARS-CoV-1, MERS-CoV, OC43, NL63 and 229E were reported elsewhere (Hoffmann et al., 2020 (link); Hoffmann et al., 2013 (link); Hofmann et al., 2005 (link); Park et al., 2016 (link); Prévost et al., 2020 (link)). Expressors of HKU1 Spike and SARS-CoV-2 S2 N-His were purchased from Sino Biological. Using the standard calcium phosphate method, 10 μg of Spike expressor and 2 μg of a green fluorescent protein (GFP) expressor (pIRES2-eGFP) was transfected into 2 × 106 293T cells. At 48 h post transfection, 293T cells were stained with CV3-1 and CV3-25 antibodies (5 μg/mL), using cross-reactive anti-SARS-CoV-1 Spike CR3022 or mouse anti-His tag (Sigma-Aldrich) as positive controls. Alexa Fluor-647-conjugated goat anti-human IgG (H+L) Abs (Invitrogen) and goat anti-mouse IgG (H+L) Abs (Invitrogen) were used as secondary antibodies. The percentage of transfected cells (GFP+ cells) was determined by gating the living cell population based on the basis of viability dye staining (Aqua Vivid, Invitrogen). Samples were acquired on a LSRII cytometer (BD Biosciences) and data analysis was performed using FlowJo v10 (Tree Star).
5
Cofilin phosphorylation analysis in HEK293T cells
6
Protein Expression in Bacterial Hosts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LB (Luria-Bertani) broth and LB agar were prepared from Difco Laboratories (USA). Mouse anti-His tag and HRP-conjugated anti-mouse IgG antibodies were purchased from Sigma (Germany). PCR master mix, as well as 1 kb DNA Ladder, was prepared from GoldBio (China). Ethanol and glacial acetic acid were purchased from Mojallali co. (Iran). Antibiotics (kanamycin and ampicillin) were purchased from Sigma (Germany). Prestained protein ladder and Isopropyl β-D-1-thiogalactopyranoside (IPTG) were purchased from SinaClon (Iran). Bacillus subtilis strain PY79 (ATCC1609) was provided by Iranian Research Organization for Science and Technology, Tehran, Iran. E. coli BL21 (DE3) star was purchased from Royan Institute, Tehran, Iran.
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?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!