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Key Product Details

Species Reactivity

Human, Mouse

Applications

Flow Cytometry, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Paraffin, Simple Western, Western Blot

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free

Concentration

1.0 mg/ml

Product Summary for Lysozyme Antibody - BSA Free

Immunogen

Full length recombinant human Lysozyme protein.

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Scientific Data Images for Lysozyme Antibody - BSA Free

Immunocytochemistry/Immunofluorescence: Lysozyme Antibody [NBP2-61118] - NIH3T3 cells were fixed and permeabilized for 10 minutes using -20C MeOH. The cells were incubated with anti-Lysozyme NBP2-61118 at 2 ug/ml overnight at 4C and detected with an anti-rabbit Dylight 488 (Green) at a 1:1000 dilution for 60 minutes. Nuclei were counterstained with DAPI (Blue). Cells were imaged using a 100X objective and digitally deconvolved.
Western Blot: Lysozyme Antibody [NBP2-61118] - Total protein from human cell lines THP-1 and HepG2, human stomach and kidney as well as mouse kidney was separated on a 4-20% gel by SDS-PAGE, transferred to 0.2 um PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 2.0 ug/ml anti-Lysozyme in 5% non-fat milk in TBST and detected with an anti-rabbit HRP secondary antibody using chemiluminescence.
Immunohistochemistry-Paraffin: Lysozyme Antibody [NBP2-61118] - Analysis of a FFPE tissue section of human kidney with Lysozyme antibody at 1:100 dilution. The staining was developed with HRP-DAB detection method and the counterstaining was performed using hematoxylin. This Lysozyme antibody generated an expected cytoplasmic staining in all the cells with strongest signal in tubular epithelial cells. Some tubules showed more of a punctate staining pattern (vesicular Lysozyme) while all of the tubules depicted a diffused signal (secreted Lysozyme). A subset of cells, especially those from glomeruli, showed nuclear positivity also.

Applications for Lysozyme Antibody - BSA Free

Application
Recommended Usage

Immunocytochemistry/ Immunofluorescence

2 ug/ml

Immunohistochemistry

1:100 - 1:250

Immunohistochemistry-Paraffin

1:100 - 1:250

Western Blot

2 ug/ml
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Published Applications

Read 8 publications using NBP2-61118 in the following applications:

Formulation, Preparation, and Storage

Purification

Immunogen affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1.0 mg/ml

Shipping

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

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: Lysozyme

Lysozyme is a ubiquitous enzyme defined as muraminidase catalyzing the hydrolysis of the beta (1-4) glycosidic bond in bacterial peptidoglycan, a major component of the bacterial cell wall. Lysozyme C in tissues and body fluids is associated with the monocyte-macrophage system and enhances the activity of immunoagents. Lysozyme C catalyzes the hydrolysis of certain mucopolysaccharides of bacterial cell walls. Specifically, it catalyzes the hydrolysis of the bacterial cell wall beta glycosidic linkages between N-acetylmuramic acid and N-acetylglucosamine. It is found in the spleen, lung, kidney, white blood cells, plasma, saliva, milk, and tears. Defects in Lysozyme C are a cause of amyloidosis type 8 (AMYL8), also known as systemic non-neuropathic amyloidosis or Ostertag-type amyloidosis.

Alternate Names

EC 3.2.1.17, lysozyme, lysozyme (renal amyloidosis)1,4-beta-N-acetylmuramidase C, lysozyme C, LZM, renal amyloidosis

Gene Symbol

LYZ

Product Documents for Lysozyme Antibody - BSA Free

Certificate of Analysis

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

Product Specific Notices for Lysozyme Antibody - BSA Free

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 Lysozyme Antibody - BSA Free (NBP2-61118):

Lysozyme Antibody: https://www.novusbio.com/products/lysozyme-antibody_nbp2-61118
Protocol for Flow Cytometry Intracellular Staining
Sample Preparation.
1. Grow cells to 60-85% confluency. Flow cytometry requires between 2 x 105 and 1 x 106 cells for optimal performance.
2. If cells are adherent, harvest gently by washing once with staining buffer and then scraping. Avoid using trypsin as this can disrupt certain epitopes of interest. If enzymatic harvest is required, use Accutase, Collagenase, or TrypLE Express for a less damaging option.
3. Reserve 100 uL for counting, then transfer cell volume into a 50 mL conical tube and centrifuge for 8 minutes at 400 RCF.
a. Count cells using a hemocytometer and a 1:1 trypan blue exclusion stain to determine cell viability before starting the flow protocol. If cells appear blue, do not proceed.
4. Re-suspend cells to a concentration of 1 x 106 cells/mL in staining buffer (NBP2-26247).
5. Aliquot out 1 mL samples in accordance with your experimental samples.

Tip: When cell surface and intracellular staining are required in the same sample, it is advisable that the cell surface staining be performed first since the fixation and permeablization steps might reduce the availability of surface antigens.

Intracellular Staining.
Tip: When performing intracellular staining, it is important to use appropriate fixation and permeabilization reagents based upon the target and its subcellular location. Generally, our Intracellular Flow Assay Kit (NBP2-29450) is a good place to start as it contains an optimized combination of reagents for intracellular staining as well as an inhibitor of intracellular protein transport (necessary if staining secreted proteins). Certain targets may require more gentle or transient permeabilization protocols such as the commonly employed methanol or saponin-based methods.
Protocol for Cytoplasmic Targets:
Optional: Perform cell surface staining as described in the previous section.
1. Fix the cells by adding 100 uL fixation solution (such as 4% PFA) to each sample for 10-15 minutes.
2. Permeabilize cells by adding 100 uL of a permeabization buffer to every 1 x 106 cells present in the sample. Mix well and incubate at room temperature for 15 minutes.
a. For cytoplasmic targets, use a gentle permeabilization solution such as 1X PBS + 0.5% Saponin or 1X PBS + 0.5% Tween-20.
b. To maintain the permeabilized state throughout your experiment, use staining buffer + 0.1% of the permeabilization reagent (i.e. 0.1% Tween-20 or 0.1% Saponin).
3. Following the 15 minute incubation, add 2 mL of the staining buffer + 0.1% permeabilizer to each sample.
4. Centrifuge for 5 minutes at 400 RCF.
5. Discard supernatant and re-suspend in 1 mL of staining buffer + 0.1% permeabilizer.
6. Stain each sample at 1 uL/ 1 x 106 cells of primary antibody or 1-3 uL/ 1 x 106 cells for directly conjugated antibodies. Mix well and incubate at room temperature for 30 minutes- 1 hour. Gently mix samples every 10-15 minutes.
7. Following the primary/conjugate incubation, add 2 mL/sample of staining buffer +0.1% permeabilizer and centrifuge for 5 minutes at 400 RCF.
8. Remove supernatant and re-suspend each sample in 2 mL staining buffer + 0.1% permeabilizer, repeat wash for 5 minutes at 400 RCF.
9. If using a directly conjugated antibody, after the second wash, re-suspend cell pellet to a final volume of 500 uL per sample and proceed with flow analysis.

Lysozyme Antibody: https://www.novusbio.com/products/lysozyme-antibody_nbp2-61118
Immunocytochemistry Protocol

Culture cells to appropriate density in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and wash the cells briefly in PBS. Add 10% formalin to the dish and fix at room temperature for 10 minutes.
2. Remove the formalin and wash the cells in PBS.
3. Permeablize the cells with 0.1% Triton X100 or other suitable detergent for 10 min.
4. Remove the permeablization buffer and wash three times for 10 minutes each in PBS. Be sure to not let the specimen dry out.
5. To block nonspecific antibody binding, incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
6. Add primary antibody at appropriate dilution and incubate overnight at 4C.
7. Remove primary antibody and replace with PBS. Wash three times for 10 minutes each.
8. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
9. Remove secondary antibody and replace with PBS. Wash three times for 10 minutes each.
10. Counter stain DNA with DAPi if required.


*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.

Lysozyme Antibody: https://www.novusbio.com/products/lysozyme-antibody_nbp2-61118
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 4 C.
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.




Lysozyme Antibody: https://www.novusbio.com/products/lysozyme-antibody_nbp2-61118
Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 10-25 ug of total protein per lane.
2. Transfer proteins to PVDF membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain the membrane with Ponceau S (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot TBS -0.05% Tween 20 (TBST).
5. Block the membrane in 5% Non-fat milk in TBST (blocking buffer) for at least 1 hour.
6. Wash the membrane in TBST three times for 10 minutes each.
7. Dilute anti-Lysozyme primary antibody in blocking buffer and incubate overnight at 4C with gentle rocking.
8. Wash the membrane in TBST three times for 10 minutes each.
9. Incubate the membrane in diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) for 1 hour at room temperature.
10. Wash the blot in TBST 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.

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