Log In Sign up
The largest database of trusted experimental protocols

H9658

Manufactured by Merck Group
Visit Supplier
Sourced in United States

H9658 is a laboratory equipment product. It is designed for use in various scientific research and testing applications. The core function of this product is to provide a specific capability or feature to support laboratory workflow and procedures. No further details about the intended use or specific applications of this product can be provided in an unbiased and factual manner.

Automatically generated - may contain errors

Lab products found in correlation

F1804, by Merck Group (4 mentions) Ab6808, by Abcam (2 mentions) Wbkls0100, by Merck Group (2 mentions) T6074, by Merck Group (2 mentions) Lipofectamine 3000 transfection reagent, by Thermo Fisher Scientific (2 mentions) Sc 10747, by Santa Cruz Biotechnology (2 mentions) A2228, by Merck Group (2 mentions) F3165, by Merck Group (2 mentions) Poly d lysine hydrobromide, by Merck Group (2 mentions) Dynabeads protein g, by Thermo Fisher Scientific (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) 4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions) Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions) A4416, by Merck Group (1 mentions) Las 4000 image analyzer, by Fujifilm (1 mentions) A2066, by Merck Group (1 mentions) Ecl western blotting detection reagent, by GE Healthcare (1 mentions) Ab56416, by Abcam (1 mentions) Sc 32233, by Santa Cruz Biotechnology (1 mentions) Las4000, by GE Healthcare (1 mentions)

43 protocols using h9658

1

Cryo-EM Imaging of Protein Localization

Cited 2 times
Check if the same lab product or an alternative is used in the 5 most similar protocols
Cells were cryo-fixed and freeze-substituted as described before (Wu et al., 2019 (link)). Epon-embedded cells were sectioned and collected on formvar-coated and carbon-evaporated copper grids. A CM12 (Philips) transmission electron microscope was used to inspect the grids. ImageJ was used for measuring the distance between membranes.
CLEM was performed for localization analysis as described previously (Knoops et al., 2015 (link)). 150-nm-thick cryo-sections were imaged with a widefield fluorescence microscope as described above. The corresponding fluorescence signals were visualized using the same filter sets as mentioned before. The grid was post-stained and embedded in a mixture containing 0.5% uranyl acetate and 0.5% methylcellulose. A CM12 transmission electron microscope under 100 kV was applied for the generation of double-tilt tomography series including a tilt range of 40° to −40° with 2.5° increments. To make CLEM images, FM and EM images were aligned using the eC-CLEM plugin (Paul-Gilloteaux et al., 2017 (link)) in Icy (http://icy.bioimageanalysis.org). The IMOD software package was used for reconstructing the tomograms.
Immuno-EM was performed as described previously (Thomas et al., 2018 (link)). Labeling of HA was performed using monoclonal antibodies (Sigma-Aldrich, H9658) followed by goat-anti-mouse antibodies conjugated to 6 nm gold (Aurion).
Krikken A.M., Wu H., de Boer R., Devos D.P., Levine T.P, & van der Klei I.J. (2020). Peroxisome retention involves Inp1-dependent peroxisome–plasma membrane contact sites in yeast. The Journal of Cell Biology, 219(10), e201906023.
+ Open protocol
+ Expand
2

Cas9 Protein Expression Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
HEK293T cells are transfected with 500 ng Cas9 and 500 ng sgRNA expressing plasmid in a 6-well plate by Lipofectamine 3000 transfection reagent (Invitrogen) according to manufacturer’s suggested protocol. 48 hours after transfection, cells are harvested and lysed with 100 μl RIPA buffer. 8ul of cell lysate is used for electrophoresis and blotting. The blots are probed with anti-HA (Sigma #H9658) and anti alpha-tubilin (Sigma #T6074) primary antibodies; then HRP conjugated anti-mouse IgG (Abcam #ab6808) and anti-rabbit IgG secondary antibodies, respectively. Visualization employed Immobilon Western Chemiluminescent HRP substrate (EMD Millipore #WBKLS0100).
Bolukbasi M.F., Gupta A., Oikemus S., Derr A.G., Garber M., Brodsky M.H., Zhu L.J, & Wolfe S.A. (2015). DNA-binding domain fusions enhance the targeting range and precision of Cas9. Nature methods, 12(12), 1150-1156.
+ Open protocol
+ Expand
3

Evaluating GroE Dependency In Vivo

Check if the same lab product or an alternative is used in the 5 most similar protocols
The experimental evaluation of GroE dependency in vivo was performed according to the previous work [26 (link)]. MGM100, a strain in which GroE expression is controlled by arabinose, was transformed with the expression vector of a candidate protein and grown in an LB medium containing 0.2% arabinose at 37 °C to log phase. The cells were then washed with fresh LB medium and subcultured in LB medium containing 1 mm diaminopimelate and 0.2% glucose for depletion (GroE– conditions) or 0.2% arabinose as a control (GroE+ conditions). Each candidate protein was expressed under leaky conditions (without IPTG induction) during cultivation. After 3 h of cultivation, the cells were harvested, suspended in lysis buffer (20 mm Tris–HCl, pH 8.0, 100 mm NaCl, and 1 mm EDTA), and disrupted by sonication. After the disruption, the uncentrifuged total fraction and the supernatant fraction obtained by 20 000 g centrifugation were separated by SDS/PAGE. The proteins in each fraction were detected by immunoblotting with an anti‐HA monoclonal antibody (SIGMA, H9658) and an anti‐mouse antibody conjugated with horseradish peroxidase (SIGMA, A4416). The chemiluminescence signal was detected by a LAS4000 image analyzer (Fujifilm).
Minami S., Niwa T., Uemura E., Koike R., Taguchi H, & Ota M. (2023). Prediction of chaperonin GroE substrates using small structural patterns of proteins. FEBS Open Bio, 13(4), 779-794.
+ Open protocol
+ Expand
4

Immunoprecipitation and Western Blot Analysis of Laforin in Fibroblasts

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
Crude extracts from primary cultures of control, EPM2A Y112X/N163D and R241X/R241X patient fibroblasts, were obtained as in (Roma-Mateo et al., 2015 (link)). One mg of proteins from the clarified extracts were immunoprecipitated as in (Sherwood et al., 2013a (link)), using anti-laforin antibody #113. Proteins in the samples were denatured using sample buffer (125 mM Tris-HCl pH 6.8,, 4% SDS, 20% glycerol, 31mg/mL DTT, 0.01% bromophenol blue) and heating to 95°C for 5 min. The samples were subjected to SDS-PAGE (12% acrylamide) and transferred onto PVDF membranes (Millipore, Madrid, Spain). Membranes were blocked with 5% skimmed milk in TBS-Tween for 1 h and slices of these membranes were incubated with the following specific antibodies: anti-HA (H9658, 1:5000; Sigma Aldrich, Madrid, Spain), anti-laforin (MABN606, 1:1000; Millipore, Madrid, Spain), anti-p62 (ab56416, 1:1000; Abcam, Cambridge, UK), anti-GAPDH (sc32233, 1:10000; Santa Cruz Biotech, USA) and anti-actin (A2066, 1:1000; Sigma Aldrich, Madrid, Spain). Thereafter, blots were washed again with TBS-Tween and further incubated for 1 h with the corresponding secondary antibody conjugated with horseradish peroxidase. Finally, membranes were washed (3×5 min) with TBS-Tween and analyzed by chemiluminiscence (ECL Western Blotting Detection Reagents, GE Healthcare, UK) using an image reader LAS-4000 (GE Healthcare, UK).
Garcia-Gimeno M.A., Rodilla-Ramirez P.N., Viana R., Salas-Puig X., Brewer M.K., Gentry M.S, & Sanz P. (2018). A novel EPM2A mutation yields a slow progression form of Lafora disease. Epilepsy research, 145, 169-177.
+ Open protocol
+ Expand
5

CFTR and COX Antibody Validation

Check if the same lab product or an alternative is used in the 5 most similar protocols
The anti-CFTR antibodies used were sc-10747, anti-CFTR (Santa Cruz Biotechnology, Dallas TX), and anti-CFTR MAB3484 (Millipore, Etobicoke, ON Canada). The COX1 antibody used was the anti-COX1 antibody EPR5866 (AB109025 Abcam Cambridge, UK), and the COX-2 antibody used was the anti-COX-2 antibody EPR12012 (AB179800 Abcam Cambridge, UK) and hemagglutinin tag (H-9658 Sigma St. Louis, MO) was used.
Carlile G.W., Yang Q., Matthes E., Liao J., Birault V., Sneddon H.F., Poole D.L., Hall C.J., Hanrahan J.W, & Thomas D.Y. (2022). The NSAID glafenine rescues class 2 CFTR mutants via cyclooxygenase 2 inhibition of the arachidonic acid pathway. Scientific Reports, 12, 4595.
+ Open protocol
+ Expand
6

eCLIP-qPCR Analysis of RNA-Protein Interactions

Cited 2 times
Check if the same lab product or an alternative is used in the 5 most similar protocols
eCLIP-qPCR was performed as reported before41 (link)–43 (link). Briefly, cells were cultured in medium with 4-thiouridine (100 μM; T4509, Sigma) for 16 h. Then cells were washed twice by cold PBS and crosslinked with ultraviolet (365 nm,150 mJ/cm2) and then lysed with NP-40 lysis buffer (FNN0021, Invitrogen) containing protease inhibitors and 1 mM dithiothreitol (P2325, Invitrogen). RNase T1 (AM2283, Invitrogen) was added to the supernatant at a final concentration of 1 U/μl and incubated at 22 °C for 15 min. Then Cyclin D1 antibody (RB-010-P, Invitrogen, dilution 1:100) or HA-tag antibody (H9658, Sigma, dilution 1:100) was added and incubated at 4 °C with rotation overnight. Forty microliters of dynabeads Protein G (10003D, Invitrogen) was added and incubated at 4 °C for 3 h. The pellets were incubated in NP-40 lysis buffer with DNase I (18047019, Invitrogen) at a concentration of 1 U/5 μl for 15 min at 37 °C. The immunoprecipitated protein–RNA complex was eluted from the beads by heat denaturing. After SDS-PAGE and nitrocellulose membrane transfer, the 35–110 KD region (a region of 75 kDa (~220 nt of RNA) above the Cyclin D1 protein size) is excised and treated with proteinase K (25530049, Invitrogen) to isolate RNA for next qPCR analysis. Primers were designed every 200 nt with 100-nt overlapping intervals to cover the full length of DILA1, listed in Table S3.
Shi Q., Li Y., Li S., Jin L., Lai H., Wu Y., Cai Z., Zhu M., Li Q., Li Y., Wang J., Liu Y., Wu Z., Song E, & Liu Q. (2020). LncRNA DILA1 inhibits Cyclin D1 degradation and contributes to tamoxifen resistance in breast cancer. Nature Communications, 11, 5513.
+ Open protocol
+ Expand
7

AHCY Protein Expression Analysis in Mice

Check if the same lab product or an alternative is used in the 5 most similar protocols
All mice were given zinc water for 10 days before any analysis. Tissue homogenates were prepared in 10 mM Tris-HCl (pH 7.5) supplemented with protease inhibitors (Roche). Twenty μg of lysate was separated by 4–12% SDS-PAGE (Invitrogen) under reducing conditions and transferred to nitrocellulose. In this study, blots were probed with two different AHCY antibodies. One antibody (ab151734, Abcam) is a rabbit polyclonal that only recognizes only human AHCY, while the other (H00000191-M07A, Abnova) is a mouse monoclonal that recognizes both human and mouse AHCY. Other antibodies used included a mouse anti-HA (H9658, Sigma), rabbit anti-hCBS [33] (link), and mouse anti-actin (A5441, Sigma). Gel images were captured and quantified using the FluorChem SP system (Alpha Innotech). The relative intensities of each protein are assigned as arbitrary units obtained by calculating the ratio of a test sample divided by a control.
Lee H.O., Wang L., Kuo Y.M., Andrews A.J., Gupta S, & Kruger W.D. (2018). S-adenosylhomocysteine hydrolase over-expression does not alter S-adenosylmethionine or S-adenosylhomocysteine levels in CBS deficient mice. Molecular Genetics and Metabolism Reports, 15, 15-21.
+ Open protocol
+ Expand
8

Protein Expression and Knockdown Protocols

Check if the same lab product or an alternative is used in the 5 most similar protocols
Antibodies against poly-PAR (4336-BPC-100, Trevigen, IF: 1:200), PARP1 (9532S, Cell Signaling Technology, WB: 1:1000), BRCA1 (D-9, Santa Cruz, IF: 1:100), HA (H9658, Sigma, WB 1:2000), γH2AX (05-636, Merck Millipore, IF: 1:1000), 53BP1 (NB100-304, Novus, IF: 1:100) and BrdU (ab6326, Abcam, IF: 1:200) were used. Full-length PARP1 was subcloned into the HA-tagged vector (pCMV-HA), the GFP-tagged vector (pFUGW) or the lentiviral vector (pHAGE-EF-Puro-DEST). The shPARP1 plasmid was constructed as described. The K23A and K418A mutant PARP1 plasmids were generated by site-directed mutagenesis and confirmed by sequencing. The shPARP1 stable cell lines and the reconstituted wild-type (WT), K23A and K418A mutant PARP1 stable cell lines were constructed according to http://www.addgene.org/protocols/plko/#C, and lentiviral packaging vectors pMD2.0G and pSPAX from NovoPro Bioscience were used.
Wang X., Mi S., Zhao M., Lu C., Jia C, & Chen Y. (2022). Quantitative Analysis of the Protein Methylome Reveals PARP1 Methylation is involved in DNA Damage Response. Frontiers in Molecular Biosciences, 9, 878646.
+ Open protocol
+ Expand
9

Western Blot Analysis of Protein Expression

Check if the same lab product or an alternative is used in the 5 most similar protocols
Three days post-transfection, cells were lysed in RIPA buffer with protease inhibitor cocktail. Samples were centrifuged at 14,000 × g for 10 min. The supernatant was harvested and quantified using BCA protein assay kit (Thermo Fisher, 23225) on Victor X5. 25 μg protein was mixed and boiled with 5 × SDS loading buffer. Samples were separated using SDS-PAGE assay. Protein was transferred to nitrocellulose membranes (Bio-Rad) for 1 hour in transfer buffer at 300 mA. The membranes were blocked at room temperature for 20 min in 5% milk-TBST and incubated with the primary antibody in 3% BSA-TBST at RT for two hours. Then the membranes were washed in TBST and incubated with secondary antibody in 3% BSA-TBST at RT for one hour and washed in TBST. Blots were visualized using Odyssey finally. The antibodies used for WB were listed below. Rabbit polyclonal anti-GAPDH (Abmart, P30008M) (1:5000 dilution), mouse monoclonal anti-HA antibody (Sigma, H9658) (1:5000 dilution), goat anti-rabbit secondary antibody (Odyssey, 926-32211) (1:5,000 dilution) and the goat anti-mouse secondary antibody (Odyssey, 926-68070) (1:5,000 dilution).
Chen Y., Liu J., Zhi S., Zheng Q., Ma W., Huang J., Liu Y., Liu D., Liang P, & Songyang Z. (2020). Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells. Nature Communications, 11, 3136.
+ Open protocol
+ Expand
10

CFTR Antibody Targeting Protocol

Check if the same lab product or an alternative is used in the 5 most similar protocols
Anti-CFTR antibodies targeting the N-terminus (sc-10747; anti-CFTR Santa Cruz Biotechnology, Dallas TX) used at 0.5 µg/ml, NBD1 (MAB3484; Millipore, Etobicoke, ON Canada) at 1 µg/ml, C-terminus (MAB3480; Millipore, and MAB25031; R&D Systems, Minneapolis, MN) and hemagglutinin tag at 1 µg/ml (H-9658 Sigma St. Louis, MO) were used.
Carlile G.W., Yang Q., Matthes E., Liao J., Radinovic S., Miyamoto C., Robert R., Hanrahan J.W, & Thomas D.Y. (2018). A novel triple combination of pharmacological chaperones improves F508del-CFTR correction. Scientific Reports, 8, 11404.
+ 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?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!