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Laminin Antibody

Catalog # NB300-144 | Novus Biologicals a Bio-Techne Brand

Key Product Details

Species Reactivity

Human, Mouse, Rat, Chinese Hamster, Invertebrate, Mammal, Rabbit, Sheep

Applications

Flow Cytometry, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry Free-Floating, Immunohistochemistry-Frozen, Immunohistochemistry-Paraffin, Western Blot

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Concentration

1 mg/ml

Product Summary for Laminin Antibody

Immunogen

Laminin Antibody was made to Laminin 111 isolated from mouse Engelbreth-Holm-Swarm (EHS) sarcoma cells. [UniProt# P19137]

Reactivity Notes

Rabbit, Fruit Bat, Chinese Hamster, and S. mansoni reactivity reported in scientific literature (PMID: 18214989, 31877588, 29251349, and 28114363 respectively). Human, Mouse, Rat, and Sheep reported in multiple pieces of scientific literature.

Localization

Secreted, Basement membrane, Extracellular matrix

Specificity

Laminin Antibody is pan-specific and reacts well with all Laminin isoforms tested: Laminin-1 (alpha-1, beta-1, and gamma-1) and Laminin-2 (alpha-2, beta-1, and gamma-1).

Marker

Basement Membrane Marker

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

337 kDa.
Disclaimer note: 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.

Scientific Data Images for Laminin Antibody

Staining of Laminin in Free Floating Rat Brain

Staining of Laminin in Free Floating Rat Brain

Immunohistological analysis of brain stem section stained with rabbit polyclonal Laminin Antibody [NB300-144], dilution 1:1,000 in red, and costained with chicken pAb to Myelin Basic Protein (MBP), dilution 1:5,000 in green. The blue is DAPI staining of nuclear DNA. Following transcardial perfusion of rat with 4% paraformaldehyde, brain was post fixed for 24 hours, cut to 45 uM, and free-floating sections were stained with the above antibodies. The laminin antibody is an excellent marker of basement membranes surrounding blood vessels, while the MBP antibody stains the myelin sheathes around axons.
Fluorescent Staining of Basement Membranes in Mouse Tissue Using Laminin Antibody

Fluorescent Staining of Basement Membranes in Mouse Tissue Using Laminin Antibody

MxIF analysis of mouse submandibular salivary gland morphogenesis. MxIF of a developmental TMA including embryonic stages (E14, E16, E18) and postnatal stages (P5 and P20) was performed using sequential application of directly conjugated antibodies to detect multiple markers of tissue structures and cell types on the same tissue sections. Tissue compartments. The epithelium, mesenchyme, neurons, and basement membranes was detected using antibodies directed towards E-cadherin (ECAD, red), platelet-derived growth factor (PDGFR, green), beta III tubulin (bIII, magenta), and Laminin Antibody [NB300-144] (LMN, cyan), respectively. Image collected and cropped by CiteAb from the following publication (http://bio.biologists.org/cgi/doi/10.1242/bio.20134309), licensed under a CC-BY license.
Strong Staining of Laminin in Basement Membranes of Blood Vessels in Free Floating Mouse Tissue

Strong Staining of Laminin in Basement Membranes of Blood Vessels in Free Floating Mouse Tissue

Staining of mouse section of cortex stained with Laminin Antibody [NB300-144] (red). Blue is DAPI staining of DNA. This antibody reveals strong staining in the basement membranes of blood vessels.

Applications for Laminin Antibody

Application
Recommended Usage

Immunocytochemistry/ Immunofluorescence

1:50-1:200

Immunohistochemistry

1:500-1:2000

Immunohistochemistry Free-Floating

1:1000-1:5000

Immunohistochemistry-Frozen

1:500-1:2000

Immunohistochemistry-Paraffin

1:500-1:2000

Western Blot

1:100-1:5000
Application Notes
This Laminin antibody detects bands at around 440, 220, and 158 kDa in Western Blot. Use in flow cytometry (PMID: 31819166) reported in scientific literature. Use in ICC/IF, IHC, IHC-Frozen, IHC-Paraffin, and Western Blot reported in multiple pieces of scientific literature. Immunostaining is enhanced by antigen retrieval with pepsin, especially paraffin tissue. br/>
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.
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Reviewed Applications

Read 9 reviews rated 4.6 using NB300-144 in the following applications:

Published Applications

Read 121 publications using NB300-144 in the following applications:

Formulation, Preparation, and Storage

Purification

IgG purified

Formulation

50% PBS, 50% glycerol

Preservative

5mM Sodium Azide

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.

Background: Laminin

Laminin, the most abundant structural and biologically active component in basement membranes, is a complex extracellular glycoprotein with high molecular weight (900 kDa). Laminin is composed of a laminin alpha (400 kDa), beta (215 kDa) and gamma chain (205 kDa), whose multidomain proteins are encoded by distinct genes (LAMA, LAMB, LAMC respectively) and have several isoforms of each chain. The biological functions of the different chains and trimer molecules are largely unknown, but some of the chains have been shown to differ with respect to their tissue distribution, reflecting diverse functions in vivo. Laminin is thought to mediate the attachment, migration and organization of cells into tissues during embryonic development by interacting with other extracellular matrix components.

Laminins are an important and biologically active part of the basal lamina, influencing cell adhesion, differentiation, migration, signaling, neurite outgrowth and metastasis, where anti-laminin antibodies can be widely used to label blood vessels and basement membranes (1). Significant quantities of laminin are found in basement membranes, the thin extracellular matrices that surround epithelial tissue, nerve, fat cells and smooth, striated and cardiac muscle. Excessive serum laminin levels have been associated with fibrosis, cirrhosis and hepatitis, serious and frequent complications of chronic active liver disease characterized by excessive deposition of various normal components of connective tissue in liver (2). Epithelial mesenchymal transition (EMT) biomarkers include fibronectin, laminin, N-cadherin, and Slug (3).

References

1. Yang, M. Y., Chiao, M. T., Lee, H. T., Chen, C. M., Yang, Y. C., Shen, C. C., & Ma, H. I. (2015). An innovative three-dimensional gelatin foam culture system for improved study of glioblastoma stem cell behavior. J Biomed Mater Res B Appl Biomater, 103(3), 618-628. doi:10.1002/jbm.b.33214

2. Mak, K. M., & Mei, R. (2017). Basement Membrane Type IV Collagen and Laminin: An Overview of Their Biology and Value as Fibrosis Biomarkers of Liver Disease. Anat Rec (Hoboken), 300(8), 1371-1390. doi:10.1002/ar.23567

3. Choi, S., Yu, J., Park, A., Dubon, M. J., Do, J., Kim, Y., . . . Park, K. S. (2019). BMP-4 enhances epithelial mesenchymal transition and cancer stem cell properties of breast cancer cells via Notch signaling. Sci Rep, 9(1), 11724. doi:10.1038/s41598-019-48190-5

Alternate Names

LAMA, Laminin A chain, laminin subunit alpha-1, laminin, alpha 1, Laminin-1 subunit alpha, Laminin-3 subunit alpha, PTBHS, S-LAM alpha, S-laminin subunit alpha

Entrez Gene IDs

284217 (Human); 16772 (Mouse); 316758 (Rat)

Gene Symbol

LAMA1

UniProt

Product Documents for Laminin Antibody

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot number in the search box below.

Product Specific Notices for Laminin Antibody

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.

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Protocols

View specific protocols for Laminin Antibody (NB300-144):

[[URL:https://www.novusbio.com/products/laminin-antibody_nb300-144]][[Caption… Antibody]]
Laminin Immunohistohemistry ABC/HRP
The fixation is routine paraformaldehyde or formalin fixation of tissue prior to paraffin embedding. However, the staining procedure must include an antigen retrieval step pretreating the deparaffinized sections with pepsin. This is required for all laminin antibodies used on paraffin tissue.
Proteolytic antigen retrieval:
Apply 250 uL of pepsin at 4mg/mL in 0.01M HCL (pH ~2.0).
Incubate for 60 minutes at 37 degrees C in a humid chamber.
Wash x2 in distilled H20, 5 min. each wash.
Mount paraffin sections on Fisher Plus slides. Bake for >2 hours at 50C.
1. Deparaffinize mounted sections in xylene: 2 changes 5 min each, and a 3rd change for 10 min.
2. Exchange solvent to ethanol with 2 changes of 100% EtOH for 5 min each.
3. Quench endogenous peroxidase for 30 min in 100% methanol + 1% H2O2.
4. Hydrate to H2O in graded ethanol series.
5 min. in 95% EtOH; 5 min. in 70% EtOH; 5 min. in running H2O. Rinse with dH2O.
Circumscribe the sections with PAP Pen.
5. Unmask antigen by proteolysis. Cover sections with 100ul of 4mg/ml of pepsin (Sigma #P6887) dissolved in 0.01M HCl. Treat for 1 hr at 37C in humidified chamber. Rinse in running H2O.
6. Blocking: Block background staining by covering sections with 100ul of PBS containing 10% normal swine serum (Blocking Buffer) for 1 hr at ambient temperature in humidified chamber.
7. Apply 1 degrees antibody. Aspirate Blocking Buffer and immediately apply rabbit anti-EHS laminin (MuirLab prep) diluted to 1 ug/ml in Blocking Buffer. Apply 100ul to sections, and incubate overnight at 4C in humidified chamber.
8. Aspirate 1 degrees antibody and wash slides in a rack by immersion in PBS with 3 changes over >=15 min.
9. Apply biotinylated 2 degrees antibody. Dilute biotinylated swine anti-rabbit 1/500 in Blocking Buffer. Apply 100ul to sections, and incubate for 2 hr. at ambient temperature in humidified chamber. (Before end of incubation, prepare ABC reagents as stated in step 10.)
10. Aspirate 2 degrees antibody and wash by immersion in PBS with 3 changes over >=15 min.
11. Apply ABC complex. Dilute Reagent A (Avidin) 1:50 in PBS + 0.1% Triton X100, mix well. In a separate tube, dilute Reagent B (Biotin-HRP) 1:50 in PBS + 0.1% Triton X100, mix well. Mix equal parts of solutions A and B. Vortex to mix well, and preincubate for at least 30 min. Immediately prior to use, dilute the 1:50 ABComplex stock an additional 1/5 (i.e., 1:250 final) with PBS + 0.1% BSA. Apply 100ul to sections, and incubate for 2 hr at ambient temperature in humidified chamber.
12. Aspirate ABC solution and Wash by immersion in PBS with 3 changes over >=30 min.
13. Develop with chromagenic substrate. Immediately before use, mix in 3 ml of PBS, 1.5 mg DAB (diaminobenzidine-[HCl]4; Sigma #D5637) and 2ul H2O2 (30%). Filter with a 0.2um syringe filter. Apply 100ul to sections and let develop for 12 min at ambient temperature. Stop chromagenic reaction by submerge slides in running H2O.
14. Dehydrate through graded alcohol series.
5 min in 70% EtOH; 5 min in 95% EtOH; 5 min in 100% EtOH; 5 min in 100% EtOH
16. Coverslip in Permount.

Additional
Immunohistochemistry Protocol De-paraffinize: xylene x2 5 min (to remove paraffin) xylene x1 10 min 100% ethanol x2 5 min (to remove xylene) Quench endogenous peroxidase: Quench with 1% H2O2 in 100% methanol (v/v) for 30 min at RT. Rehydrate: 95% ethanol x1 5 min 70% ethanol x1 5 min distilled H2O x2 5 min Circumscribe tissue sections with PAP pen. Proteolytic antigen retrieval: Apply 250 ul of pepsin at 4 mg/ml in 0.01M HCl (pH ~2.0). Incubate for 60 min at 37C in a humid chamber. Wash x2 in distilled H2O, 5 min each wash. Block background: Apply 250 ul of 10% normal goat serum in PBS. Incubate for 30 min at 37C. Pour off excess blocking solution from slides, do not allow tissue to dry. Immunostaining - primary antibody: A. Apply 250 ul of anti-laminin 1 degrees Ab at 1:1000 in PBS containing 10% goat serum. B. Apply 250 ul of 10% goat serum in PBS as negative control. Incubate overnight at 37C in a humid chamber. Immunostaining - secondary antibody: Wash x2 in PBS, 5 min each wash. Apply 250 ul of 2 degrees Ab at 1:500 in PBS. Incubate for 30 min at 37C in a humid chamber. Wash x2 in PBS, 5 min each wash. DAB substrate: Apply 250 ul DAB solution and allow brown color to develop for 30 min at RT. DAB is carcinogenic therefore dispose of it as hazardous chemical waste. Rinse briefly in running distilled H2O to stop reaction. Wash x2 in distilled H2O, 5 min each wash. Mount: Air dry slides for a few minutes. Apply 3-4 drops of Crystal/Mount to tissue sections. Spread evenly by rotation. Dry slides in a 37C oven for 1-2 hours. RECIPES FOR LAMININ STAINING PROTOCOL 10X PBS Stock Solution 1X PBS - Working Solution 1.37M NaCl 80.06 g 137 mM NaCl 0.027M KCl 2.01 g 2.7 mM KCl 0.043M Na2HPO4 6.11 g 4.3 mM Na2HPO4 0.014M KH2PO4 1.92 g 1.4 mM KH2PO4 Dissolve in 800 ml distilled H2O. 100 ml stock: 900 ml distilled H2O. pH to 7.4 with 5N NaOH. QS to 1L with distilled H2O. The following volumes are for 20 tissue sections (18 test and 2 controls). Pepsin Solution Dissolve 20 mg of pepsin in 5 ml of 0.01M HCl (pH ~2.0). Pepsin: Roche 03 117 901 001 (from porcine stomach) (EC 3.4.23.1)) Endogenous Peroxidase Block 1% (v/v) = 2.5 ml of 30% H2O2 in 250 ml of 100% methanol (where 30% H2O2 is treated as 100%). Non-specific Protein Block Prepare a 10% solution by diluting 1 ml of normal goat serum in 9 ml of PBS. Goat serum: Sigma G-9023. 1 degrees and 2 degrees Antibodies 1 degrees Ab: rabbit anti-rat laminin PAb (Novus Biologicals NB 300-144). Prepare at 1:1000 by adding 4.5 ul 1 degrees Ab to 4.5 ml of 10% goat serum in PBS. 2 degrees Ab: goat anti-rabbit IgG-HRP (Santa Cruz sc-2030). Prepare at 1:500 by adding 10 ul 2 degrees Ab to 5 ml of PBS. DAB substrate: DAB: Sigma D-4293. DAB (3,3' diaminobenzidine) is carcinogenic. Prepare by dissolving one DAB tablet and one H2O2 tablet in 5 ml of distilled H2O. Counterstain: Counterstain is not recommended for laminin IHC. Mount: Crystal/Mount, an aqueous based, mounting medium, is from Biomeda (catalog no. M02). LAMININ IMMUNOHISTOCHEMISTRY-HRP PROTOCOL (formalin-fixed paraffin-embedded rat liver sections) (Novus Biologicals NB 300-144) This last protocol is from the lab of: Thomas F. Tracy, Jr., M.D. Professor of Surgery and Pediatrics Vice Chairman, Department of Surgery Brown Medical School Pediatric Surgeon-in-Chief Hasbro Children's Hospital Room 147 593 Eddy Street Providence, RI 02903
[[URL:https://www.novusbio.com/products/laminin-antibody_nb300-144]][[Caption… Antibody]]
Western Blot
1. SDS-PAGE on 5% mini-gel under reducing conditions
2. Electroblot to nitrocellulose by Towbin methods
3. Remove nitrocellulose sheet from electroblotting sandwich and rinse briefly in dH2O.
4. Fixation: In a glass dish immerse the blot in 25% isopropanol/10% acetic acid/ 65% dH2O. Cover and shake gently for at least 30 min. at room temperature.
3. Remove the blot from fixative and wash in a large volume changes of dH2O for > 10 min.
4. Place blot in plastic tray with lid. Equilibrate >10 min. with Washing Buffer. Pour off.
5. Blocking: Place the blot in Blocking Buffer (just enough to cover). Incubate with gentle shaking for at least 1 h (overnight if background is a big problem). Pour off.
6. Primary Antibody: Dilute PcAbLN antibody (3/4 1 ug/ml) in Blocking Buffer. Add just enough to cover blot and incubate with shaking for 2 h at 37C or overnight at room temp.
7. Pour off primary antibody and wash X3 with Washing Buffer over 20-30 min (or more).
8. Peroxidase-conjugated Secondary Antibody: Dilute peroxidase conjugated 2 degrees IgG (Dako, affinity purified) diluted 1:2000 in Blocking Buffer. Add just enough to cover the blot and incubate with shaking for 2 h at 37C.
9. Wash blot thoroughly (30 min and up to hours) in Washing Buffer and then with a final wash in Washing buffer without Triton.
10. Chemiluminescent Detection: According to manufacturers instructions.


Washing Buffer
0.05 Tris-HCl, pH 7.4
1.5% NaCl
0.1% Triton X100

Blocking Buffer
Washing Buffer
5% powered milk
(dissolve for hours, filter)

FAQs for Laminin Antibody

Showing  1 - 3 of 3 FAQs Showing All
  • Q: I have a question concerning the antibody NB300-144. The antibody blocks the function of the laminin. Can you tell me, which site of the laminin the antibody attacks? Because I need an antibody which block the binding from laminin to the integrin receptor by binding to the globular domain (integrin binding site) of the laminin.

    A: Our bioassays show that this antibody binds to laminin and inhibits most, if not all, of its cell adhesion and growth promotive properties. In culture, the antibody hinders cell migration on a laminin substratum. It also inhibits neurite and cell process outgrowth. Applied to tissue sections, it inhibits neurite outgrowth by neurons seeded onto the tissue section. As this is a polyclonal antibody, it is nearly impossible to determine the specific antigen binding sites. No molecular level tests for laminin domain recognition have been performed. However, based on bioassays, it appears that the mix of antibodies in this purified polyclonal anti-serum bind multiply across the large laminin molecule to an extent that its biological activities are inhibited; that is, cell receptor binding is markedly hindered. It is the best, among many, laminin function-blocking preps we have tested. Most polyclonal antibodies raised in rabbit against a mouse laminin-1 will inhibit the activity of many rodent laminin isoforms. This one is especially specific, avid and purified for that purpose.

  • Q: Do you have any WB images tested by NB300-144 on human? I see the WB image on the datasheet, but I do not know what sample is loaded on each lane?

    A: The samples used for the western blot image are Laminin-1 (LN-1) protein from EHS tumor and Laminin-2 (LN-2) protein from heart.

  • Q: What isoforms does this product react with?

    A: Laminin Antibody [NB300-144] is pan-specific and reacts well with all Laminin isoforms tested: Laminin-1 (alpha-1, beta-1, and gamma-1) and Laminin-2 (alpha-2, beta-1, and gamma-1).

Showing  1 - 3 of 3 FAQs Showing All
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