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Ab3298

Manufactured by Abcam
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Ab3298 is an Antibody product from Abcam. It is a primary antibody that can be used for various research applications.

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Lab products found in correlation

Quantity one 4, by Bio-Rad (1 mentions) Ab150113, by Abcam (1 mentions) Lsm 710 confocal microscope, by Zeiss (1 mentions) Ab150077, by Abcam (1 mentions) Mab1585, by Merck Group (1 mentions) Alexa fluor coupled secondary antibody, by Thermo Fisher Scientific (1 mentions) Hcs studio cell analysis software, by Thermo Fisher Scientific (1 mentions) Lactate dehydrogenase a ldha, by Cell Signaling Technology (1 mentions) Cleaved parp asp214, by Cell Signaling Technology (1 mentions) Stat3, by Cell Signaling Technology (1 mentions) Anti β actin antibody, by Merck Group (1 mentions) Cleaved caspase 3 asp175, by Cell Signaling Technology (1 mentions) Ab7291, by Abcam (1 mentions) G7295, by Merck Group (1 mentions) Mtc02, by Abcam (1 mentions) Gm130, by Merck Group (1 mentions) Hek293t, by American Type Culture Collection (1 mentions) A21207, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Panc 1, by American Type Culture Collection (1 mentions)

16 protocols using ab3298

1

Fibroblast Immunofluorescence for PMPCA

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Cultured patient fibroblasts were transferred onto chambered slides and grown to 50%–60% confluence. The fibroblasts were fixed with 4% paraformaldehyde for 20 min and 10% Triton-X was used to permeabilize the cells before staining. The fixed slides were stored and used up to a week. Rabbit anti-PMPCA from Novus Biologicals (SAB1303187; 100 µL) made in rabbit was used as a primary antibody at a ratio of 1:100 overnight at 4°C. Anti-mitochondrial antibody (mouse) from Abcam (ab 3298) was used for costaining at 1:200 overnight at 4°C.
Abcam frataxin antibody (mouse) (17A11) (ab113691) was also used at 1:200 with Novus PMPCA for colocalization studies.
Joshi M., Anselm I., Shi J., Bale T.A., Towne M., Schmitz-Abe K., Crowley L., Giani F.C., Kazerounian S., Markianos K., Lidov H.G., Folkerth R., Sankaran V.G, & Agrawal P.B. (2016). Mutations in the substrate binding glycine-rich loop of the mitochondrial processing peptidase-α protein (PMPCA) cause a severe mitochondrial disease. Cold Spring Harbor Molecular Case Studies, 2(3), a000786.
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2

Quantifying Mitochondrial Protein Levels

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Western blot was performed to study the level of PMPCA protein on the fibroblasts available from the patient. Fibroblasts from two control human cell lines (ATCC-2127 and ATCC-2104) were used as controls. Anti-PMPCA antibody (NBP1-89126, 1:100 dilution, Novus Biologicals) was used to probe the specific protein band at 58 kDa. The results of the study were visualized using enhanced chemiluminescence using the program Quantity One 4.2.1 (Bio-Rad) and were compared with appropriate age- and tissue-matched controls normalized for the loading control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or β-tubulin.
To identify the probable interactions of PMPCA with other proteins that may be processed in the mitochondria, we also performed quantitative protein analysis for CO4I1, frataxin, PINK1, and a mitochondrial antibody cocktail (ab3298, Abcam).
Joshi M., Anselm I., Shi J., Bale T.A., Towne M., Schmitz-Abe K., Crowley L., Giani F.C., Kazerounian S., Markianos K., Lidov H.G., Folkerth R., Sankaran V.G, & Agrawal P.B. (2016). Mutations in the substrate binding glycine-rich loop of the mitochondrial processing peptidase-α protein (PMPCA) cause a severe mitochondrial disease. Cold Spring Harbor Molecular Case Studies, 2(3), a000786.
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3

Immunofluorescent Staining of Hippocampus

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Immunofluorescent staining was performed using manufacturer recommended protocols. Brains stored in paraformaldehyde were cryoprotected in sucrose and formaldehyde solution and then sectioned using a sliding microtome. Coronal 50-μm sections of dorsal hippocampus were mounted on gelled slides. Slides underwent an antigen retrieval protocol in 10 mM citrate buffer pH 6.0 for 40 min at 60°C. The sections were washed in phosphate buffered saline (PBS) plus Tween (pH 7.4) six times for 5 min each at room temperature. After incubation in 5% normal goat serum in PBS+tween for 2 h, sections were incubated in primary antibodies (mouse monoclonal anti-mitochondria (ab3298, Abcam) dilution 1 : 500, mouse monoclonal ERα (MA1-27107, ThermoScientific) dilution 1 : 200, rabbit polyclonal ERβ (PA5-16476, ThermoScientific) dilution 1 : 200) for 48 h at 4°C. Again, the sections were washed with PBS, 6 times for 5 min each before secondary antibody incubation (goat anti-rabbit IgG AF488 (ab150077, Abcam) dilution 1 : 1000, goat anti-mouse IgG AF488 (ab150113, Abcam) dilution 1 : 1000) for 4 h at room temperature. After a final series of rinses (PBS 6 times for 5 min each), slides were coverslipped using fluoromount G containing a DAPI counterstain. Slides were imaged on a LSM710 confocal microscope (Carl Zeiss International) and digital images processed as stated above.
Brooks S.W., Dykes A.C, & Schreurs B.G. (2017). A High-Cholesterol Diet Increases 27-Hydroxycholesterol and Modifies Estrogen Receptor Expression and Neurodegeneration in Rabbit Hippocampus. Journal of Alzheimer's disease : JAD, 56(1), 185-196.
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4

Immunodetection of Retinal and Brain Cells

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Immunodetection in flat mounts and brain coronal sections was carried out as reported (Galindo-Romero et al., 2011 (link); Nadal-Nicolás et al., 2015 (link)). Primary antibodies were: mouse anti-Brn3a (1:500; MAB1585, Merck Millipore; Madrid, Spain), mouse anti-human mitochondria (1:800, ab3298 Abcam, Cambridge, United Kingdom), and rabbit anti-melanopsin (1:1,000; AB-N39 Advanced Targeting Systems ATS, Joure, Netherlands). Secondary detection was carried out with Alexa Fluor-coupled secondary antibodies (1:500; Molecular Probes; Thermo Fisher Scientific, Madrid, Spain). Retinal whole-mounts and brain coronal sections were mounted with anti-fading mounting media.
Norte-Muñoz M., Lucas-Ruiz F., Gallego-Ortega A., García-Bernal D., Valiente-Soriano F.J., de la Villa P., Vidal-Sanz M, & Agudo-Barriuso M. (2021). Neuroprotection and Axonal Regeneration Induced by Bone Marrow Mesenchymal Stromal Cells Depend on the Type of Transplant. Frontiers in Cell and Developmental Biology, 9, 772223.
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5

Quantifying Mitochondrial Morphology via High-Content Imaging

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Cells were fixed with 4% paraformaldehyde for 10 min at room temperature, then permeabilized, blocked with 0.5% bovine serum albumin, and incubated with primary antibodies (each 1:500 dilution) for 1 h at room temperature, followed by incubation with Cy3- or Alexa Fluor 488-conjugated secondary antibody for 1 h. DNA was counterstained with 0.5 μg/mL DAPI.
For high-content imaging analysis, images were obtained and analyzed using a CellInsight cellomics platform with HCS studio cell analysis software (Thermo Fisher Scientific). The Spot Detector BioApplication was used to quantify the total fluorescence intensity of mitochondrial signals and to count the number of nucleoli in each cell. Each cell was defined by a DAPI channel.
RNAi screening was described previously [12 (link)]. Briefly, 2,500 IMR-90 cells were seeded with 5 nM each siRNA in 96-well plates. After 3 days, cells were fixed and subjected to immunofluorescence using an anti-mitochondrial antibody (ab3298, Abcam). Sixteen images per well were taken using CellInsight with 20× magnification. The total area of mitochondria/cell was calculated using the Spot Detector BioApplication. Screens were performed in three biological replicates and hits were defined by the magnitude of change in the mitochondrial area, with statistical analysis using Student’s t-test between control siRNA and samples.
Igata T., Tanaka H., Etoh K., Hong S., Tani N., Koga T, & Nakao M. (2022). Loss of the transcription repressor ZHX3 induces senescence-associated gene expression and mitochondrial-nucleolar activation. PLoS ONE, 17(1), e0262488.
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6

Antibody Detection for Cancer Research

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Antibodies for phospho-CDK9 (#2549), CDK7 (#2916), Survivin (#2808), phospho-H2A.X (Ser139; #9718), Cleaved Caspase-3 (Asp175; #9661), Cleaved PARP (Asp214; #5625), MGMT (#2739), Myeloid Cell Leukemia 1 (Mcl-1) (#5453), HK2 (#2867), Pyruvate kinase isoform M2 (PKM2) (#4053), Lactate dehydrogenase A (LDHA) (#3582), and Stat-3 (#9139) were purchased from Cell Signaling Technology. Anti–b-actin antibody was purchased from Sigma-Aldrich. Antibodies for CDK9 (sc-484), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (sc-25778), b-actin (sc-47778), and p53 (sc-126) were purchased from Santa Cruz Biotechnology. Anti–x-linked inhibitor of apoptosis (XIAP) (NB100–56183) antibody was purchased from Novus Biologicals. Anti-cytochrome c (ab13575), Phospho-RNA Pol II CTD (ab5095), and mitochondria (ab3298) antibodies were purchased from Abcam. TG02 is licensed and was provided by Tragara Pharmaceuticals. TMZ was purchased from Sigma-Aldrich.
Su Y.T., Chen R., Wang H., Song H., Zhang Q., Chen L.Y., Lappin H., Vasconcelos G., Lita A., Maric D., Li A., Celiku O., Zhang W., Meetze K., Estok T., Larion M., Abu-Asab M., Zhuang Z., Yang C., Gilbert M.R, & Wu J. (2017). Novel Targeting of Transcription and Metabolism in Glioblastoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(5), 1124-1137.
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7

Characterization of CLPTM1L Protein Localization

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The human embryonic kidney cell line HEK293T, human pancreatic cancer cell line PANC-1, and mouse kidney cell line IMCD3 (all from ATCC) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Mediatech Inc, Herndon, VA) supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY).
Commercial antibodies used included those for endogenous CLPTM1L (HPA014791, Sigma, St. Louis, MO), FLAG-tagged CLPTM1L (M2 F1804, Sigma), the endoplasmic reticulum (ER) marker Calnexin, (C4731, Sigma), a mitochondrial marker (MTC02, ab3298, Abcam Cambridge, MA), the centrosome marker gamma-tubulin (T5192, Sigma), the Golgi marker GM130 (G7295, Sigma), alpha-tubulin (ab7291, Abcam) and Myosin-9/MYH10 (sc-33729, Santa Cruz Biotechnology, Santa Cruz, California). Secondary antibodies were Alexa Fluor 594® or 488® donkey anti-mouse or anti-rabbit IgG (H+L) (A21202, A21203, A21206 and A21207, Life Technologies).
Jia J., Bosley A.D., Thompson A., Hoskins J.W., Cheuk A., Collins I., Parikh H., Xiao Z., Ylaya K., Dzyadyk M., Cozen W., Hernandez B.Y., Lynch C.F., Loncarek J., Altekruse S.F., Zhang L., Westlake C.J., Factor V.M., Thorgeirsson S., Bamlet W.R., Hewitt S.M., Petersen G.M., Andresson T, & Amundadottir L.T. (2014). CLPTM1L promotes growth and enhances aneuploidy in pancreatic cancer cells. Cancer research, 74(10), 2785-2795.
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8

Mitochondrial Content and MDR1 Expression

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Tissue microarrays (TMAs) of the 50 resected lesions were constructed utilizing 1.0-mm cores in triplicate from the same sample when possible. Antibodies for assessing mitochondrial content (Abcam; ab3298; diluted 1:750) and MDR1 expression (Abcam; ab170903; diluted 1:250) were acquired from commercial sources. A 45-min antigen retrieval (HTTR) step was performed prior to incubation with either primary antibody at room temperature for 45 min. Detection of immunolabeling was performed using anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies, and counterstaining was performed with 3,3′-diaminobenzidine. The mitochondrial stain was scored on a 4-point scale based on the percentage of cells with strong granular cytoplasmic staining: 0 (0–5%), 1+ (5–25%), 2+ (25–50%), and 3+ (50–100%). Similarly, the MDR1 stain was scored on a 4-point scale based on the percentage of cells with strong membranous staining: 0 (0–5%), 1+ (5–25%), 2+ (25–50%), and 3+ (50–100%). Both of these were scored in a blinded manner with respect to tumor type by an expert urologic pathologist (ASB). A “normalized” index of mitochondrial staining was calculated by subtracting the MDR1 staining scores from the mitochondrial staining scores (i.e., subtractive normalization).
Rowe S.P., Gorin M.A., Solnes L.B., Ball M.W., Choudhary A., Pierorazio P.M., Epstein J.I., Javadi M.S., Allaf M.E, & Baras A.S. (2017). Correlation of 99mTc-sestamibi uptake in renal masses with mitochondrial content and multi-drug resistance pump expression. EJNMMI Research, 7, 80.
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9

Mitochondrial Morphology Analysis in Cells

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To obtain high resolution pictures for analysis of mitochondrial morphology HEK293 cells and primary human dermal fibroblasts were co-stained with antibodies against MTC02 (Abcam, ab3298; 1:500) and α-Tubulin (Abcam, ab52866, 1:500) overnight at 4°C. To increase detection of SIRT4-eGFP fusion proteins (in the case of stably transfected HEK293 cells) primary antibodies against GFP (Nacalai Tesque, Inc., GF090R, 1:1000) were employed. Secondary antibodies were Alexa Fluor 488-conjugated goat anti-rat, Alexa Fluor 546-conjugated goat anti-mouse IgG, and Alexa Fluor 633-conjugated goat anti-rabbit IgG. Acquisitions were performed with the UltraVIEW spinning disk confocal microscope (Perkin Elmer) with emission wavelengths of 468 nm, 488 nm, 543 nm, and 633 nm and the Volocity 6.3 software (Perkin Elmer). Pictures were further analyzed using ImageJ software v1.49k employing specific macros for the analysis of mitochondrial mass and mitochondrial tube formation/length analysis (Suppl. Material & Methods).
Lang A., Anand R., Altinoluk-Hambüchen S., Ezzahoini H., Stefanski A., Iram A., Bergmann L., Urbach J., Böhler P., Hänsel J., Franke M., Stühler K., Krutmann J., Scheller J., Stork B., Reichert A.S, & Piekorz R.P. (2017). SIRT4 interacts with OPA1 and regulates mitochondrial quality control and mitophagy. Aging (Albany NY), 9(10), 2163-2189.
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10

Immunohistochemical Detection of Mitochondria

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The tissues were fixed by submersion in neutral-buffered formalin (Sigma-Aldrich, Schnelldorf, Germany) for 24 h, and then trimmed and placed in histological cassettes for dehydration, inclusion in paraffin (Sigma-Aldrich) and staining with hematoxylin and eosin (Merck KGaA, Darmstadt, Germany). The tissues were subjected to an indirect immunoperoxidase method as follows: Antigen retrieval was achieved by exposing the samples to 90°C for 30 min in citrate buffer (10 mM, pH 6.0; Sigma-Aldrich), followed by overnight incubation at 4°C with the mouse monoclonal anti-human mitochondria antibody (ab3298; Abcam, Cambridge, MA, USA). Next, the samples were treated using a streptavidin-biotin detection method (using a Histostain-Plus Bulk Kit, a LAB-SA Detection System and a DAB-Plus Reagent Set; Invitrogen Life Technologies, Camarillo, CA, USA) followed by hematoxylin counterstaining. The images of the tissues were obtained with a DM3000 laboratory microscope (Leica Microsystems, Inc., Buffalo Grove, IL, USA).
CIFUENTES F.F., VALENZUELA R.H., CONTRERAS H.R, & CASTELLÓN E.A. (2015). Development of an orthotopic model of human metastatic prostate cancer in the NOD-SCIDγ mouse (Mus musculus) anterior prostate. Oncology Letters, 10(4), 2142-2148.
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