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Goat anti mouse igg f ab 2

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Goat anti-mouse IgG F(ab')2 is a secondary antibody preparation that specifically recognizes the F(ab')2 fragment of mouse immunoglobulin G (IgG) antibodies. It is produced by immunizing goats with mouse IgG and isolating the F(ab')2 portion of the resulting antibodies.

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Lab products found in correlation

Glutamax, by Thermo Fisher Scientific (4 mentions) Goat anti mouse igm f ab 2, by Jackson ImmunoResearch (3 mentions) Cd43 microbeads, by Thermo Fisher Scientific (2 mentions) Cd38 90 biotin, by Thermo Fisher Scientific (2 mentions) Cd11c n418 biotin, by Thermo Fisher Scientific (2 mentions) Goat anti mouse κ f ab 2, by Southern Biotech (2 mentions) Mouse rbaff, by R&D Systems (2 mentions) Rat anti mouse cd40 1c10, by Thermo Fisher Scientific (2 mentions) Mouse ril 4, by Thermo Fisher Scientific (2 mentions) 2 mercaptoethanol, by Thermo Fisher Scientific (2 mentions) Cd43 microbead, by Miltenyi Biotec (2 mentions) Anti biotin microbead, by Miltenyi Biotec (2 mentions) Rpmi 1640, by Corning (2 mentions) Mem non essential amino acid, by Corning (2 mentions) Ovalbumin (ova), by Merck Group (2 mentions) Mouse anti mouse ubiquitin p4d1, by Santa Cruz Biotechnology (2 mentions) Donkey anti rabbit igg alexa 488, by Thermo Fisher Scientific (2 mentions) Goat anti rabbit igg alexa 546, by Thermo Fisher Scientific (2 mentions) Donkey anti rat igg alexa546, by Thermo Fisher Scientific (2 mentions) Donkey anti rat alexa647, by Thermo Fisher Scientific (2 mentions)

8 protocols using goat anti mouse igg f ab 2

1

Isolation and Culture of Germinal Center B Cells

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B splenocytes were MACS-purified, collecting the negative fraction of CD43 Microbeads (Miltenyi Biotec). GC B cells were MACS-purified, collecting the negative fraction of CD43 Microbeads, CD38 (90) Biotin (Thermo Fisher Scientific), CD11c (N418) Biotin (Thermo Fisher Scientific) and anti-Biotin Microbeads (Miltenyi Biotec). Cells were cultured in RPMI 1640 (Corning) supplemented with 10% FBS (MilliporeSigma and Thermo Fisher Scientific), 1x Penicillin-Streptomycin, 1x MEM Nonessential Amino Acids (Corning), 1mM Sodium Pyruvate, 2mM GlutaMax, and 55 μM 2-Mercaptoethanol (Thermo Fisher Scientific). Cells were stimulated with the following reagents: 10μg/ml goat anti-mouse IgM F(ab’)2, 10μg/ml goat anti-mouse IgG F(ab’)2 (Jackson ImmunoResearch Labs), 10μg/ml goat anti-mouse κ F(ab’)2 (SouthernBiotech), 10ng/ml mouse rBaff (R&D Systems), 5μg/ml rat anti-mouse CD40 (1C10) and 10ng/ml mouse rIL-4 (Thermo Fisher Scientific). For in vitro-derived GC B cell cultures, B splenocytes were cultured on 40LB cells (3T3 fibroblasts expressing CD40L and secreting BAFF) with addition of 1ng/ml mouse rIL-4.
Ramezani-Rad P., Chen C., Zhu Z, & Rickert R.C. (2020). Cyclin D3 Governs Clonal Expansion of Dark Zone Germinal Center B Cells. Cell reports, 33(7), 108403.
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2

Measuring Calcium Flux in iNK Cells

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iNK cells were stained with Indo-1 AM (Thermo Fisher) per the manufacturer’s protocol and co-stained with CD56 and a fixable live/dead indicator dye (Invitrogen). For stimulation through CD16, cells were pre-coated with an anti-CD16 antibody (3G8; BioLegend). Cells were run for 30 seconds on a FACS Fortessa X-30 (BD Biosciences) to obtain baseline measurements of free and bound calcium by measuring the shift in emission spectra. Then, goat anti-mouse IgG f(ab’)2 (Jackson ImmunoResearch Laboratories) was added as a crosslinking agent, and cells were immediately returned to the flow cytometer. Data was collected for an additional 4 minutes. For inonomycin stimulation, non-antibody coated iNK cells were first run unstimulated for 30 seconds followed by addition of ionomycin to achieve a final concentration of 1 μM, then run for an additional 4 minutes. Calcium flux was calculated based on the ratio of free and bound calcium over time.
Woan K.V., Kim H., Bjordahl R., Davis Z.B., Gaidarova S., Goulding J., Hancock B., Mahmood S., Abujarour R., Wang H., Tuininga K., Zhang B., Wu C.Y., Kodal B., Khaw M., Bendzick L., Rogers P., Ge M.Q., Bonello G., Meza M., Felices M., Huffman J., Dailey T., Lee T.T., Walcheck B., Malmberg K.J., Blazar B.R., Bryceson Y.T., Valamehr B., Miller J.S, & Cichocki F. (2021). Harnessing Features of Adaptive NK Cells to Generate iPSC-Derived NK Cells for Enhanced Immunotherapy. Cell stem cell.
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3

Immunofluorescence Analysis of Proteasome Components

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We used Rat anti-mouse LAMP1 (BD Bioscience, San Jose, CA), Rabbit anti-mouse α-Tubulin (Abcam), Rabbit anti-mouse γ-Tubulin (Abcam), Rabbit anti-mouse S4/19S RP (Abcam), Rabbit anti-mouse αβ/20S proteasome (Abcam), Rabbit anti-mouse α4/20S proteasome (Abcam), Mouse anti-mouse Ubiquitin P4D1 (Santa Cruz), Rabbit anti-mouse pSyk (Y525/526) (cellsignalling), Rabbit anti-mouse Syk (Abcam), anti-mouse β-actin (Abcam), Rat anti-mouse CD45R (BD Bioscience), Goat anti-mouse IgGFab2 (Jackson ImmunoResearch), Goat anti-mouse IgM Fab2 (Jackson ImmunoResearch). For secondary antibodies: Donkey anti-rabbit IgG-Alexa488 (LifeTech), Goat anti-rabbit IgG-Alexa546 (ThermoScientific), Donkey anti-rat IgG-Alexa546 (ThermoScientific), Donkey anti-rat-Alexa647 (ThermoScientific), Phalloidin-Alexa647 (ThermoScientific), DAPI (Abcam). Ovalbumin was purchased from Sigma-Aldrich, MG-132 and Epoxomicin were purchased from Merk (Millipore).
Ibañez-Vega J., Del Valle Batalla F., Saez J.J., Soza A, & Yuseff M.I. (2019). Proteasome Dependent Actin Remodeling Facilitates Antigen Extraction at the Immune Synapse of B Cells. Frontiers in Immunology, 10, 225.
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4

Proteasome and Cytoskeletal Dynamics

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We used rat anti-mouse LAMP1 (BD Bioscience, #553792, 1:200), rabbit anti-mouse α-Tubulin (Abcam, ab#6160, 1:200), rabbit anti-acetyl-α-Tubulin (Lys40; cell signaling, #5335, 1:200), rabbit anti-mouse γ-Tubulin (Abcam, #Ab11317, 1:1000), rabbit anti-mouse S4/19S RP (Abcam, #Ab223765, 1:100), rabbit anti-mouse αβ/20S proteasome (Abcam, #Ab22673, 1:200), anti-mouse Ecm29 (Abcam, #Ab28666, 1:100), mouse anti-mouse Ubiquitin P4D1 (Santa Cruz, #Sc-8017, 1:1000), anti-mouse anti-actin (cloneC4, ImmunO, #691001), rabbit anti-mouse Arp2 (Cellsignal, #5614, 1:500), goat anti-mouse IgGFab2 (Jackson ImmunoResearch), rabbit anti-OVA (Sigma-Aldrich, #C6534, 1:500). For secondary antibodies: donkey anti-rabbit IgG-Alexa488 (LifeTech, 1:200), goat anti-rabbit IgG-Alexa546 (ThermoScientific, 1:200), Donkey anti-rat IgG-Alexa546 (ThermoScientific, 1:200), Donkey anti-rat-Alexa647 (ThermoScientific, 1:200), Phalloidin-Alexa-647 (Life Technology, #22287, 1:200), DAPI (Abcam). Ovalbumin and Nocodazole were purchased from Sigma-Aldrich, MG-132, and Epoxomicin were purchased from Merk (Millipore). Bsc2118-FL-Bodipy was kindly provided by Ulrike Kuckelkorn (Mlynarczuk-Bialy et al., 2014 (link)).
Ibañez-Vega J., Del Valle F., Sáez J.J., Guzman F., Diaz J., Soza A, & Yuseff M.I. (2021). Ecm29-Dependent Proteasome Localization Regulates Cytoskeleton Remodeling at the Immune Synapse. Frontiers in Cell and Developmental Biology, 9, 650817.
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5

Isolation and Culture of Germinal Center B Cells

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B splenocytes were MACS-purified, collecting the negative fraction of CD43 Microbeads (Miltenyi Biotec). GC B cells were MACS-purified, collecting the negative fraction of CD43 Microbeads, CD38 (90) Biotin (Thermo Fisher Scientific), CD11c (N418) Biotin (Thermo Fisher Scientific) and anti-Biotin Microbeads (Miltenyi Biotec). Cells were cultured in RPMI 1640 (Corning) supplemented with 10% FBS (MilliporeSigma and Thermo Fisher Scientific), 1x Penicillin-Streptomycin, 1x MEM Nonessential Amino Acids (Corning), 1mM Sodium Pyruvate, 2mM GlutaMax, and 55 μM 2-Mercaptoethanol (Thermo Fisher Scientific). Cells were stimulated with the following reagents: 10μg/ml goat anti-mouse IgM F(ab’)2, 10μg/ml goat anti-mouse IgG F(ab’)2 (Jackson ImmunoResearch Labs), 10μg/ml goat anti-mouse κ F(ab’)2 (SouthernBiotech), 10ng/ml mouse rBaff (R&D Systems), 5μg/ml rat anti-mouse CD40 (1C10) and 10ng/ml mouse rIL-4 (Thermo Fisher Scientific). For in vitro-derived GC B cell cultures, B splenocytes were cultured on 40LB cells (3T3 fibroblasts expressing CD40L and secreting BAFF) with addition of 1ng/ml mouse rIL-4.
Ramezani-Rad P., Chen C., Zhu Z, & Rickert R.C. (2020). Cyclin D3 Governs Clonal Expansion of Dark Zone Germinal Center B Cells. Cell reports, 33(7), 108403.
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6

Antibody Characterization for Cell Analysis

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Anti-CD98 clone UM7F8, mIgG1 control antibody clone MOPC21, anti-mouse cKit clone ACK45 and anti-human NGFR clone C40–1457 were purchased from BD. Anti-Mouse CD90.1 Clone OX-7, anti-CD98 clone MEM108, and anti-mouse CD98 Clone RL388, were purchased from Biolegend. Anti-CD98 H300 was purchased from Santa Cruz Biotechnology. Anti-Ubiquitin clone P4D1 and anti-Met clone L41G3 were purchased from CST. Goat Anti-human IgG F(ab’)2, Goat Anti-mouse IgG F(ab’)2, and Goat anti-mouse IgG F(ab’) were purchased from Jackson. Humanized anti-human MET (LY2875358/Emibetuzumab) and non-cross competing MET antibody (LSN2148068) have been described previously and were provided by Eli Lilly(13 (link)). LSN2148068 was conjugated to DyLight650 using a DyLight microscale conjugation kit (Life Technologies) according to the manufacturer’s directions. Secondary reagents for western blot (Goat anti-mouse IgG-800, Goat anti-mouse IgG-680, Goat anti-rabbit IgG-800 and goat anti-rabbit IgG-680 were obtained from Licor.
Ablack J.N., Ortiz J., Bajaj J., Trinh K., Lagarrigue F., Cantor J.M., Reya T, & Ginsberg M.H. (2020). MARCH Proteins Mediate Responses to Anti-tumor Antibodies. Journal of immunology (Baltimore, Md. : 1950), 205(10), 2883-2892.
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7

Nectin-4 and Nectin-1 Expression Analysis

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Confluent monolayers of cells were detached from 75 cm2 tissue culture flasks using Accutase cell dissociation buffer (Innovative Cell Technologies, San Diego, CA), washed, and labeled with mouse IgG, a mAb against Nectin-4 (MAB2659, clone #337516) or a mAb against Nectin-1 (MAB2880, clone #610835) using 2.5 μg mAb/106 cells in Flow Buffer [phosphate buffered saline (PBS) containing 2.5% newborn calf serum and 0.02% sodium azide] for 30 min at 4°C [18 (link)]. Cells were washed and incubated with goat anti-mouse IgG F(ab’)2 (Jackson ImmunoResearch, West Grove, PA), then washed again and incubated with streptavidin-allophycocyanin conjugate (Jackson ImmunoResearch, West Grove, PA) for 30 min each. Cells were washed and fixed in Flow Buffer containing 1% formaldehyde and analyzed in the University of Minnesota Flow Cytometry Resource with a BD Accuri™ C6 flow cytometer (BD Biosciences, San Jose, CA) using the Accuri™ software.
Boylan K.L., Buchanan P.C., Manion R.D., Shukla D.M., Braumberger K., Bruggemeyer C, & Skubitz A.P. (2016). The expression of Nectin-4 on the surface of ovarian cancer cells alters their ability to adhere, migrate, aggregate, and proliferate. Oncotarget, 8(6), 9717-9738.
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8

Multiparametric Flow Cytometry for CAR19 and CD34

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Flow cytometry (BD LSRII) and cell sorting (FACSAria III) were performed using routine T cell panels (BD Biosciences Multitest 6 color BTNK antibodies) with the addition of goat anti-mouse IgG F(ab)2 (Jackson Immunoresearch) for CAR19 detection and mouse anti-human Qbend10 (Miltenyi Biotec) for human CD34 staining of RQR8.
Qasim W., Zhan H., Samarasinghe S., Adams S., Amrolia P., Stafford S., Butler K., Rivat C., Wright G., Somana K., Ghorashian S., Pinner D., Ahsan G., Gilmour K., Lucchini G., Inglott S., Mifsud W., Chiesa R., Peggs K.S., Chan L., Farzeneh F., Thrasher A.J., Vora A., Pule M, & Veys P. (2017). Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. Science translational medicine, 9(374).
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