Product Datasheet - Novus Biologicals

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antibody, CD11b, with, Antibody, buffer, wash, Western, [PMID:, Wash, minutes., room, times, minutes, three, temperature., Remove, sections, each., from, membrane, using, blocking, incubate, Updated, 1/19/2017, v.20.1, solution, Mouse), Page, model

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Product Datasheet
CD11b Antibody
NB110-89474
Unit Size: 0.1 ml
Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.
Reviews: 3 Publications: 24
Protocols, Publications, Related Products, Reviews, Research Tools and Images at:
www.novusbio.com/NB110-89474
Updated 1/19/2017 v.20.1
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NB110-89474
CD11b Antibody
Product Information
Unit Size 0.1 ml
Concentration 1.00 mg/ml
Storage Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.
Clonality Polyclonal
Preservative 0.02% Sodium Azide
Purity Immunogen affinity purified
Buffer PBS
Target Molecular Weight 127 kDa
Product Description
Host Rabbit
Gene ID 3684
Gene Symbol ITGAM
Species Human, Mouse, Rat
Reactivity Notes We have feedback that this antibody does not work in human samples with
Western blot. Immunogen has 100% homology to bovine and 88% homology
with sheep.
Marker Microglia Marker, Myeloid Marker
Immunogen A synthetic peptide made to an internal region (within residues 250-350) of the
mouse CD11b protein. [Swiss-Prot# P05555]
Product Application Details
Applications Western Blot, Simple Western, Flow Cytometry,
Immunocytochemistry/Immunofluorescence, Immunohistochemistry,
Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin
Recommended Dilutions Western Blot 2 ug/ml, Simple Western 1:50, Flow Cytometry 1:10-1:1000,
Immunohistochemistry 1:400, Immunocytochemistry/Immunofluorescence 1:200,
Immunohistochemistry-Paraffin 1:400, Immunohistochemistry-Frozen
Application Notes This CD11b antibody is useful for Western blot analysis, Immunocytochemistry,
Flow Cytometry (PMID 21422470) and IHC-paraffin embedded sections. In
Western blot a specific band is observed ~ 160 kDa and an apparant nonspecific band is observed ~ 56 kDa. Prior to immunostaining paraffin tissues,
antigen retrieval with sodium citrate buffer (pH 6.0) is recommended. In ICC/IF,
membrane staining was observed in Raw 264.7 cells. This antibody does not
appear to work in human with Western blot. Use in ImmunohistochemistryFrozen reported in scientific literature (PMID 23980916) The observed molecular
weight of the protein may vary from the listed predicted molecular weight due to
post translational modifications, post translation cleavages, relative charges, and
other experimental factors.
Page 1 of 6 v.20.1 Updated 1/19/2017
Images
Western Blot: CD11b Antibody [NB110-89474] - Detection of CD11b in
RAW 264.7 whole cell lysates using NB110-89474.
Immunocytochemistry/Immunofluorescence: CD11b Antibody [NB11089474] - Staining of mouse pancreas using CD11b antibody at 1:400
dilution. Nuclei counterstained with DAPI (blue). Image provided by
product review by verified customer.
Immunohistochemistry: CD11b Antibody [NB110-89474] - Analysis of
CD11b in human renal cancer using DAB with hematoxylin counterstain.
Immunocytochemistry/Immunofluorescence: CD11b Antibody [NB11089474] - CD11b antibody was tested in Raw264.7 cells with Dylight 488
(green). Nuclei and alpha-tubulin were counterstained with DAPI (blue)
and Dylight 550 (red).
Page 2 of 6 v.20.1 Updated 1/19/2017
Simple Western: CD11b Antibody [NB110-89474] - Simple Western lane
view shows a specific band for Cd11b in 1.0 mg/ml of Dentate Gyrus
from Rat Brain at an antibody concentration of 1:50. This experiment was
performed under reducing conditions using the 12-230 kDa separation
system. Image provided through customer review.
Publications
Xu X, Meng Q, Erben U et al. Myeloid-derived suppressor cells promote B-cell production of IgA in a TNFR2dependent manner. Cell. Mol. Immunol. May 2 2016 12:00AM [PMID: 27133471]
Kim D, Kim TH, Wu G et al. Extracellular Release of CD11b by TLR9 Stimulation in Macrophages PLoS ONE Mar 9
2016 12:00AM [PMID: 26954233] (WB, Mouse)
Yang X, Scott HA, Monickaraj F et al. Basement membrane stiffening promotes retinal endothelial activation
associated with diabetes. FASEB J 2016 Feb [PMID: 26443820]
Makar TK, Nimmagadda VKC, Singh IS et al. TrkB agonist, 7, 8-dihydroxyflavone, reduces the clinical and
pathological severity of a murine model of multiple sclerosis. J Neuroimmunol. 2016 Jan 06 [PMID: 26943953] (IHCP, Mouse)
Algieri F, Rodriguez-Nogales A, Garrido-Mesa J et al. Intestinal anti-inflammatory activity of calcium pyruvate in the
TNBS model of rat colitis: Comparison with ethyl pyruvate. Biochem. Pharmacol. 2016 Jan 13 [PMID: 26774455]
(IHC-P, Rat)
Details:
CD11b antibody was used for IHC-P analysis of colonic tissues from trinitrobenzenesulfonic acid /TNBS model of
colitis in Rats which received or not the treatment of ethyl pyruvate /EP, calcium pyruvate monohydrate /CPM or
sulphasalazine/SAZ (Fig. 2).
Makar TK, Gerzanich V, Nimmagadda VK et al. Silencing of Abcc8 or inhibition of newly upregulated Sur1-Trpm4
reduce inflammation and disease progression in experimental autoimmune encephalomyelitis. J Neuroinflammation.
2015 Nov 20 [PMID: 26581714] (IHC-P, Mouse)
Ulasov I, Borovjagin AV, Kaverina N et al. MT1-MMP silencing by an shRNA-armed glioma-targeted conditionally
replicative adenovirus CRAd) improves its anti-glioma efficacy in vitro and in vivo. Cancer Lett 2015 Jun 4 [PMID:
26052095]
Details:
Using the Alexa Fluor (R) 488 conjugated version of NB110-89474, catalog number NB110-89474AF488.
Stiess M, Wegehingel S, Nguyen C et al. A Dual SILAC Proteomic Labeling Strategy for Quantifying Constitutive and
Cell-Cell Induced Protein Secretion J. Proteome Res. 2015 Jul 20 [PMID: 26189946] (ICC/IF, Rat)
Details:
Integrin alpha M/CD11b antibody used for Immunofluorescence analysis at a dilution of 1:200 on Rat forebrain glia
cells.
Sawano S, Suzuki T, Do MK et al. Supplementary immunocytochemistry of hepatocyte growth factor production in
activated macrophages early in muscle regeneration. Anim Sci J 2014 Dec [PMID: 25185534]
Ohshima R, Hotsumi K. Age-related decrease in glucagon-like peptide-1 in mouse prefrontal cortex but not in
hippocampus despite the preservation of its receptor American Journal of BioScience . 2015-02-29 (IHC-Fr, Mouse)
Page 3 of 6 v.20.1 Updated 1/19/2017
de la Pisa IG, Cebrian C, Ortega JE et al. Cytokine pathway disruption in a mouse model of schizophrenia induced by
Munc18-1a overexpression in the brain. J Neuroinflammation 2014 Jul 29 [PMID: 25069615] (WB, Mouse)
Details:
CD11b antibody used for WB application at 1:1000 dilution in mouse model of schizophrenia induced by Munc18-1a
overexpression/OE in the brain (see full text for sample preparation details). Figures 3a and 4a shows the WB of
microglia /macrophage marker CD11bas single band of 160 kDa in cerebral cortex and striatum tissues of Munc18OE and wild-type mice.
Sakaguchi S, Shono JI, Suzuki T et al. Implication of anti-inflammatory macrophages in regenerative motoneuritogenesis: Promotion of myoblast migration and neural chemorepellent semaphorin 3A expression in injured
muscle Int. J. Biochem. Cell Biol. 2014 Jun 02 [PMID: 24886696] (IHC-Fr, ICC/IF, Mouse)
Details:
Antibody used for ICC-IF (as macrophage marke) on macrophages isolated from peritoneal cavity of male C57BL/6
mice (Figure 1A, B). Antibody also used for IHC-Fr on tibialis anterior muscle of mice for detecting the total population
of activated monocytes/macrophages (Figure 6B, C)
More publications at http://www.novusbio.com/NB110-89474
Page 4 of 6 v.20.1 Updated 1/19/2017
Procedures
Western Blot protocol specific for CD11b Antibody (NB110-89474)
Western Blot Protocol
1. Perform SDS-PAGE on samples to be analyzed, loading 40 ug of total protein per lane.
2. Transfer proteins to membrane according to the instructions provided by the manufacturer of the
membrane and transfer apparatus.
3. Stain according to standard Ponceau S procedure (or similar product) to assess transfer success,
and mark molecular weight standards where appropriate.
4. Rinse the blot.
5. Block the membrane using standard blocking buffer for at least 1 hour.
6. Wash the membrane in wash buffer three times for 10 minutes each.
7. Dilute primary antibody in blocking buffer and incubate 1 hour at room temperature.
8. Wash the membrane in wash buffer three times for 10 minutes each.
9. Apply the diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturers
instructions) and incubate 1 hour at room temperature.
10. Wash the blot in wash buffer three times for 10 minutes each (this step can be repeated as
required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturers instructions.
**Note: Tween-20 can be added to the blocking or antibody dilution buffer at a final concentration of
0.05-0.2%.
Immunohistochemistry-Paraffin Embedded Sections (NB110-89474)
Immunohistochemistry-Paraffin Embedded Sections
Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling
temperature for 10 minutes. Cool slides on bench-top for 30 minutes.
Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in wash buffer for 5 minutes.
3. Block each section with 100-400 ul blocking solution for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul diluted primary antibody. Incubate overnight at 4C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul biotinylated diluted secondary antibody. Incubate 30 minutes at room temperature.
7. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes
each.
8. Add 100-400 ul Streptavidin-HRP reagent to each section and incubate for 30 minutes at room
temperature.
9. Wash sections three times in wash buffer for 5 minutes each.
10. Add 100-400 ul DAB substrate to each section and monitor staining closely.
11. As soon as the sections develop, immerse slides in deionized water.
12. Counterstain sections in hematoxylin.
13. Wash sections in deionized water two times for 5 minutes each.
14. Dehydrate sections.
15. Mount coverslips.
Immunocytochemistry/Immunofluorescence Protocol for CD11b Antibody (NB110-89474)
Immunocytochemistry Protocol
Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.
1. Remove culture medium and add 10% formalin to the dish. Fix at room temperature for 30 minutes.
2. Remove the formalin and add ice cold methanol. Incubate for 5-10 minutes.
3. Remove methanol and add washing solution (i.e. PBS). Be sure to not let the specimen dry out.
Wash three times for 10 minutes.
4. To block nonspecific antibody binding incubate in 10% normal goat serum from 1 hour to overnight
at room temperature.
5. Add primary antibody at appropriate dilution and incubate at room temperature from 2 hours to
overnight at room temperature.
6. Remove primary antibody and replace with washing solution. Wash three times for 10 minutes.
7. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
8. Remove antibody and replace with wash solution, then wash for 10 minutes. Add Hoechst 33258 to
wash solution at 1:25,0000 and incubate for 10 minutes. Wash a third time for 10 minutes.
9. Cells can be viewed directly after washing. The plates can also be stored in PBS containing Azide
covered in Parafilm (TM). Cells can also be cover-slipped using Fluoromount, with appropriate sealing.
*The above information is only intended as a guide. The researcher should determine what protocol
best meets their needs. Please follow safe laboratory procedures.
Page 5 of 6 v.20.1 Updated 1/19/2017
Limitations
This product is for research use only and is not approved for use in humans or in clinical diagnosis.
Primary Antibodies are guaranteed for 1 year from date of receipt.
For more information on our 100% guarantee, please visit www.novusbio.com/guarantee
Earn gift cards/discounts by submitting a review: www.novusbio.com/reviews/submit/NB110-89474
Earn gift cards/discounts by submitting a publication using this product:
www.novusbio.com/publications
Novus Biologicals USA
8100 Southpark Way, A-8
Littleton, CO 80120
USA
Phone: 303.730.1950
Toll Free: 1.888.506.6887
Fax: 303.730.1966
novus@novusbio.com
Novus Biologicals Canada
461 North Service Road West, Unit B37
Oakville, ON L6M 2V5
Canada
Phone: 905.827.6400
Toll Free: 855.668.8722
Fax: 905.827.6402
canada@novusbio.com
Products Related to NB110-89474
NBP1-30158 Raw 264.7 Whole Cell Lysate
HAF008 Goat anti-Rabbit IgG Secondary Antibody [HRP (Horseradish
Peroxidase)]
NBP1-75297 Goat anti-Rabbit IgG (H+L) Secondary Antibody [HRP]
NB110-89474AF488
Novus Biologicals Europe
19 Barton Lane
Abingdon Science Park
Abingdon, OX14 3NB, United Kingdom
Phone: (44) (0) 1235 529449
0800 37 34 15
Fax: (44) (0) 1235 533420
info@bio-techne.com
General Contact Information
www.novusbio.com
Technical Support: technical@novusbio.com
Orders: orders@novusbio.com
General: novus@novusbio.com

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