Imagequant las 4000 machine

Manufactured by GE Healthcare

Sourced in United States

The ImageQuant LAS 4000 is a digital imaging system designed to capture and analyze chemiluminescent, fluorescent, and colorimetric signals from various types of gel and blot-based assays. The system features a high-resolution camera, multiple excitation and emission filter options, and advanced image analysis software, enabling accurate quantification of protein and nucleic acid samples.

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ImageQuant LAS 4000 (1851 citations) ImageQuant (354 citations) ImageQuant LAS 4000 mini machine (10 citations)

18 protocols using imagequant las 4000 machine

APE1 Expression in Activated B Cells

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Naive, DZ and LZ B cells were isolated from human tonsils by flow
cytometry. One fraction of naïve B cells was stimulated in
vitro for 72h with IL-21 (10ng/mL, #130–094-563, Miltenyi
Biotec) and CD40L (1μg/mL, #310–02, PeproTech). Total protein
extraction was performed using RIPA buffer (#89901, Thermo Fisher) supplemented
with phosphatase and protease inhibitor (#4906845001 and #11873580001, Roche).
10μg of whole-cell extracts from each cell subset were separated by
SDS-PAGE (12% w/v), blotted onto nitro-cellulose membranes and incubated with
anti-APE1 antibody (sc-17774, Santa Cruz Biotechnology). β-actin was used
as a loading control (#A5441, Sigma-Aldrich). Enhanced chemiluminescence (ECL)
development was performed after incubation with secondary antibodies conjugated
to horseradish peroxidase (HRP) using Clarity Western ECL Substrate (#1705061,
Bio-Rad) according to manufacturer’s instructions. Images were acquired
on an Image Quant LAS 4000 machine (GE Healthcare Life Sciences). Densitometry
analysis was performed using Image Studio software version 5.2.5 (LI-COR
Biosciences).

Roco J.A., Mesin L., Binder S.C., Nefzger C., Gonzalez-Figueroa P., Canete P.F., Ellyard J., Shen Q., Robert P.A., Cappello J., Vohra H., Zhang Y., Nowosad C.R., Schiepers A., Corcoran L.M., Toellner K.M., Polo J., Meyer-Hermann M., Victora G, & Vinuesa C.G. (2019). Class Switch Recombination Occurs Infrequently in Germinal Centers. Immunity, 51(2), 337-350.e7.

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SDS-PAGE and Immunoblotting Protocol

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Cells were harvested in buffer containing 2% SDS, 10% glycerol, and 50 mM Tris-HCl, and immediately boiled for 10 min. Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fischer Scientific # 23225). DTT (100 mM) and bromophenol blue was added to samples which were run on a 12% SDS-polyacrylamide gel. Blotting was performed on nitrocellulose membranes (GE Healthcare 10600003) which were then stained with Ponceau S. Dry milk (5%) in phosphate buffered saline with 0.1% tween-20 (PBS-T) was used for both blocking of membranes and dilution of secondary- and primary antibodies. Blocking was performed for 30 min at room temperature, incubation with primary antibody at 4°C overnight, and incubation with secondary antibody for 1 h at room temperature. Membranes were washed minimum three times in PBS-T both before addition of secondary antibody, and again before development using a Chemiluminicent Peroxidase substrate kit (Sigma-Aldrich, CPS3500) and an ImageQuant LAS 4000 machine (GE Healthcare).

Hoem G., Bowitz Larsen K., Øvervatn A., Brech A., Lamark T., Sjøttem E, & Johansen T. (2019). The FMRpolyGlycine Protein Mediates Aggregate Formation and Toxicity Independent of the CGG mRNA Hairpin in a Cellular Model for FXTAS. Frontiers in Genetics, 10, 249.

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Western Blot Analysis of P. falciparum Gametocyte Proteins

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P. falciparum NF54 gametocyte extract was prepared as previously described.⁴³ (link) The extract was mixed with NuPAGE™ LDS sample buffer and heated for 15 minutes as 56°C. To reduce samples, a final concentration of 25mM dithiothreitol was added. The proteins were separated on a NuPAGE™ 4-12% Bis-Tris 2D-well gel and subsequently transferred to a 0.22 μm nitrocellulose membrane (Bio-Rad) using the Trans-Blot Turbo system (Bio-Rad). The blots were cut into strips, blocked with 5% skimmed milk in PBS and incubated with 5 μg/ml monoclonal antibody. The strips were subsequently incubated with 1:5,000 anti-human IgG-HRP (Pierce, Cat. No 31412) in PBST. Clarity Western ECL substrate (Bio-Rad) was used for development and strips were imaged with the ImageQuant LAS4000 machine (GE Healthcare).

Fabra-García A., Hailemariam S., de Jong R.M., Janssen K., Teelen K., van de Vegte-Bolmer M., van Gemert G.J., Ivanochko D., Semesi A., McLeod B., Vos M.W., de Bruijni M.H., Bolscher J.M., Szabat M., Vogt S., Kraft L., Duncan S., Kamya M.R., Feeney M.E., Jagannathan P., Greenhouse B., Dechering K.J., Sauerwein R.W., King C.R., MacGill R.S., Bousema T., Julien J.P, & Jore M.M. (2023). Highly potent, naturally acquired human monoclonal antibodies against Pfs48/45 block Plasmodium falciparum transmission to mosquitoes. Immunity, 56(2), 406-419.e7.

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Protein Expression Analysis in Brain Tissue

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Tissues were lysed using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s instructions. Following RNA extraction and cDNA synthesis, quantitative PCR (qPCR) was performed in triplicate on a CFX96 Real-Time PCR machine (Bio-Rad). Primer sequences are presented in SI. Glut1, phospho-AMPK, and p53 protein levels were assessed by Western blots using antibodies detailed in SI and procedures previously described (12 (link)). Protein bands were visualized on an ImageQuant LAS 4000 machine (NA-931, GE Healthcare) using the ECL Detection Kit (RPN 2109, GE Healthcare). Band intensities were determined using ImageJ software (NIH). Brain parenchymal and microvasculature fractions were separated using a modified version of a protocol described by Galea et al. (39 (link)). Briefly, fractions were separated on an 18% dextran gradient and either processed immediately or stored at –80°C until required. For details, see SI.

Tang M., Park S.H., Petri S., Yu H., Rueda C.B., Abel E.D., Kim C.Y., Hillman E.M., Li F., Lee Y., Ding L., Jagadish S., Frankel W.N., De Vivo D.C, & Monani U.R. (2021). An early endothelial cell–specific requirement for Glut1 is revealed in Glut1 deficiency syndrome model mice. JCI Insight, 6(3), e145789.

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Deyolking and Lysis of Zebrafish Embryos

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Heat shocked and dechorionated embryos were collected at 3dpf. To deyolk embryos, a borosilicate injection needle was used to mechanically disrupt yolks. Embryos were next washed in deyolking buffer without calcium (Link et al., 2006 (link)), spun at 300rcf and washed in wash buffer (110mM NaCl, 3.5mM KCl, 2.7mM CaCl₂, 10mM Tris/Cl) containing Complete Mini protease inhibitor mixture (Roche Diagnostics). Deyolked embryos were lysed with modified LeMeer’s Lysis Buffer (50mM Tris pH 7.5, 150mM NaCl, 1mM EDTA, 1% IGEPAL, 0.1% sodium deoxycholate) supplemented with Complete Mini protease inhibitor cocktail (Lemeer et al., 2007 (link)) before being centrifuged at low speed and sonicated by a Sonic Dismembranator Model 300 (Fisher Scientific). Using manufacturer’s protocol, protein samples were gel electrophoresed using 4–12% Bis-Tris gel and transferred onto PVDF membrane (Invitrogen NuPage system). Blots were incubated 1:5000 anti-myc (abcam ab9106), followed by 1:5000 horse anti-mouse HRP secondary (Cell Signaling Technology 7076). Blots were imaged via the Super Signal West Femto visualization system (Life Sciences) on an ImageQuant LAS4000 machine (GE Life Sciences). Following imaging of myc antibody labeling, blots were stripped for 15 minutes in Restore Western Blot Stripping Buffer (Thermo Scientific 21059) and reprobed with 1:5000 anti-actin (calbiochem cp01).

Hanovice N.J., McMains E, & Gross J.M. (2016). A GAL4-inducible transgenic toolkit for the in vivo modulation of Rho GTPase activity in zebrafish. Developmental dynamics : an official publication of the American Association of Anatomists, 245(8), 844-853.

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Quantification of MAPK and AKT Phosphorylation

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The phosphorylation of p38 MAPK and AKT were determined as previously described for ERK1/2 phosphorylation [53 (link)] using western blot analysis. Quantification was performed using an ImageQuant Las-4000 machine (GE Healthcare).

Toledano S., Sabag A.D., Ilan N., Liburkin-Dan T., Kessler O, & Neufeld G. (2023). Plexin-A2 enables the proliferation and the development of tumors from glioblastoma derived cells. Cell Death & Disease, 14(1), 41.

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Quantification of XBP1 splicing

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Synthesized cDNA as described above was used to measure levels of unspliced and spliced XBP1. mRNA was measured by regular PCR performed with GoTaq Green Master Mix (Promega cat. no. M7123) on a T100 Thermo Cycler (Bio-Rad, cat. no. 186-1096). The primers targeting the spliced XBP1 region (116 bp) (SI Appendix, Table S4) were designed according to a previous study (46 (link)). PCR products were resolved by agarose gel electrophoresis and visualized using GelGreen stain (Biotium, cat. no. 41005) on an ImageQuant LAS 4000 machine (GE Healthcare). Densitometric analysis of product bands was performed using the Gel Analyzer function in Fiji from NIH (47 (link)).

Zhao G., Tan Y., Cardenas H., Vayngart D., Wang Y., Huang H., Keathley R., Wei J.J., Ferreira C.R., Orsulic S., Cheng J.X, & Matei D. (2022). Ovarian cancer cell fate regulation by the dynamics between saturated and unsaturated fatty acids. Proceedings of the National Academy of Sciences of the United States of America, 119(41), e2203480119.

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Protein Extraction and Western Blot Analysis

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The proteins were extracted using lysis buffer [10 mM Tris-HCl (pH 7.5), 1 mM ethylene-diaminetetraacetic acid, 10% glycerol, 0.5% NP-40 detergent, 400 mM NaCl, 4 µg/ml of aprotinin, phenylmethylsulfonyl fluoride and dithiothreitol]. The BCA protein assay was used for the protein determination. Total protein (10 µg loaded per lane) was separated using SDS-PAGE (on a 10% polyacrylamide gel), at 300 mA for 90 min and transferred on a nitrocellulose membrane (Invitrogen; Thermo Fisher Scientific, Inc.). Blocking of the membrane was performed using 4% skimmed milk for 60 min at room temperature. The protein expression levels of CPA4, E-cadherin and β-actin were assessed using western blot analysis. These proteins were detected using specific antibodies to CPA4 (1:200; HPA021030; Sigma-Aldrich; Merck KGaA), E-cadherin (1:1,000; M106; Takara Bio, Inc.), and β-actin (1:1,000; #3700; Cell Signaling Technology, Inc.). The membrane was incubated in primary antibodies for overnight at 4°C. β-actin served as a loading control. ECL anti-mouse or anti-rabbit IgG, peroxidase-linked whole antibody was used for secondary antibody (GE Healthcare Life Sciences, Little Chalfont, UK). The signals were detected using the ECL Western Blotting Detection System and an Image Quant LAS 4000 machine (GE Healthcare Life Sciences).

Handa T., Katayama A., Yokobori T., Yamane A., Fujii T., Obayashi S., Kurozumi S., Kawabata-Iwakawa R., Gombodorj N., Nishiyama M., Asao T., shirabe K., Kuwano H, & Oyama T. (2019). Carboxypeptidase A4 accumulation is associated with an aggressive phenotype and poor prognosis in triple-negative breast cancer. International Journal of Oncology, 54(3), 833-844.

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Western Blot Analysis of DNA Damage Markers

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Proteins were resolved by Mini Gel SDS-PAGE (Bio-Rad system) and transferred to nitrocellulose membrane (GE Healthcare) as previously described^{30 (link)}. All the blocking and antibody incubations were done in TBS −0.2% Tween-20 5% BSA (Fisher). The following primary antibodies were used: anti-53BP1 (1:1000, Millipore), anti-ATM (1:1000, Abcam), anti-Chk1-phS317 (1:1000, Cell Signalling Technology), anti-GFP (1:1000), anti-HA (1:1000, Sigma), anti-H2AX-P (1:1000, Abcam), anti-p53 (1:1000, DO-1, SantaCruz), anti-p53-phS15 (1:1000, NEB), anti-RNaseH1 (1:1000, Abcam), anti-TetR (1:1000, TETO2, MoBiTec), anti-tubulin (1:5000, Abcam), and appropriate HRP-conjugated secondary antibodies were used: anti-mouse (1:10000, Cell Signalling Technology), anti-rabbit (1:10000, Cell Signalling Technology) and anti-rat (1:10000, Abcam). Immuno-reactive bands were detected by chemoluminescence induced by Supersignal reagent and detected with the ImageQuant LAS 4000 machine (GE Healthcare). Quantification was performed using ImageJ.

Brustel J., Kozik Z., Gromak N., Savic V, & Sweet S.M. (2018). Large XPF-dependent deletions following misrepair of a DNA double strand break are prevented by the RNA:DNA helicase Senataxin. Scientific Reports, 8, 3850.

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Western Blot Detection of BgGRN

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Western blot detection of BgGRN was done using 250 ng protein suspended in Laemmli protein loading buffer. Samples were heated at 95 °C for 10 min, then loaded on 10% (vol/vol) SDS/PAGE gels and run on the Mini PROTEAN Tetra system (Bio-Rad, Hercules, California, USA) at 200 V and 180 mA. Samples were then blotted for 2.5 h onto 0.45 μm supported nitrocellulose membranes (Bio-Rad). Blocking was done for 1 h at room temperature in 5% (wt/vol) skimmed milk prepared in Tris-buffered saline (TBS) solution plus 0.1% Tween-20 (TBS-T buffer) before staining for 1 h in anti-V5 mouse IgG (recombinant BgGRN) or mouse anti-BgGRN IgG (native BgGRN) primary antibody at a concentration of 1:5000 in blocking buffer. Membranes were washed in TBS-T buffer for 10 min, then twice for 5 min each and once in TBS solution for 5 min. Membranes were then stained for 1 h in HRP-conjugated rabbit anti-mouse IgG antibody diluted 1:5000 in blocking buffer followed by a wash step as de- scribed earlier. Detection was accomplished by incubating the membranes in Super Signal West Dura Extended Duration substrate (Thermo Fisher Scientific). Chemiluminescent signals were acquired on the ImageQuant LAS 4000 machine (GE Healthcare).

Hambrook J.R., Gharamah A.A., Pila E.A., Hussein S, & Hanington P.C. (2019). Biomphalaria glabrata Granulin Increases Resistance to Schistosoma mansoni Infection in Several Biomphalaria Species and Induces the Production of Reactive Oxygen Species by Haemocytes. Genes, 11(1), 38.

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