製品の概要

  • 製品名
    Anti-Lipocalin-2 / NGAL antibody
    Lipocalin-2 / NGAL 一次抗体 製品一覧
  • 製品の詳細
    Rabbit polyclonal to Lipocalin-2 / NGAL
  • 由来種
    Rabbit
  • 特異性
    A customer has submitted a positive Abreview for rat samples in IHC. However, customers have experienced problems with rat samples and this is likely due to the low homology between the immunogen and the rat protein (30%). Some batches of this antibody may work in rat, but this is not batch tested.
  • アプリケーション
    適用あり: WB, IHC-P, ICC/IFmore details
  • 種交差性
    交差種: Human
  • 免疫原

    Synthetic peptide corresponding to Human Lipocalin-2/ NGAL aa 50-150 conjugated to Keyhole Limpet Haemocyanin (KLH).
    (Peptide available as ab41759, ab41759, ab41759, ab41759, ab41758, ab41758, ab41758, ab41758, ab41758)

  • ポジティブ・コントロール
    • Recombinant Human Lipocalin-2 / NGAL protein (ab95007) can be used as a positive control in WB. ab41105 gave a positive result in Human Colon Tissue Lysate.

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab41105 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

アプリケーション Abreviews 特記事項
WB Use a concentration of 1 µg/ml. Detects a band of approximately 21 kDa (predicted molecular weight: 23 kDa).
IHC-P 1/250 - 1/1000.

 

ICC/IF Use a concentration of 5 µg/ml.

ターゲット情報

  • 機能
    Iron-trafficking protein involved in multiple processes such as apoptosis, innate immunity and renal development. Binds iron through association with 2,5-dihydroxybenzoic acid (2,5-DHBA), a siderophore that shares structural similarities with bacterial enterobactin, and delivers or removes iron from the cell, depending on the context. Iron-bound form (holo-24p3) is internalized following binding to the SLC22A17 (24p3R) receptor, leading to release of iron and subsequent increase of intracellular iron concentration. In contrast, association of the iron-free form (apo-24p3) with the SLC22A17 (24p3R) receptor is followed by association with an intracellular siderophore, iron chelation and iron transfer to the extracellular medium, thereby reducing intracellular iron concentration. Involved in apoptosis due to interleukin-3 (IL3) deprivation: iron-loaded form increases intracellular iron concentration without promoting apoptosis, while iron-free form decreases intracellular iron levels, inducing expression of the proapoptotic protein BCL2L11/BIM, resulting in apoptosis. Involved in innate immunity, possibly by sequestrating iron, leading to limit bacterial growth.
  • 組織特異性
    Expressed in bone marrow and in tissues that are prone to exposure to microorganism. High expression is found in bone marrow as well as in uterus, prostate, salivary gland, stomach, appendix, colon, trachea and lung. Not found in the small intestine or peripheral blood leukocytes.
  • 配列類似性
    Belongs to the calycin superfamily. Lipocalin family.
  • 細胞内局在
    Secreted. Upon binding to the SLC22A17 (24p3R) receptor, it is internalized.
  • Information by UniProt
  • 参照データベース
  • 別名
    • 24p3 antibody
    • 25 kDa alpha 2 microglobulin related subunit of MMP9 antibody
    • 25 kDa alpha-2-microglobulin-related subunit of MMP-9 antibody
    • Alpha 2 microglobulin related protein antibody
    • HGNC:6526 antibody
    • HNL antibody
    • Lcn 2 antibody
    • Lcn2 antibody
    • Lipocalin-2 antibody
    • Migration stimulating factor inhibitor antibody
    • MSFI antibody
    • Neutrophil gelatinase associated lipocalin antibody
    • Neutrophil gelatinase associated lipocalin precursor antibody
    • Neutrophil gelatinase-associated lipocalin antibody
    • NGAL antibody
    • NGAL_HUMAN antibody
    • Oncogene 24p3 antibody
    • Oncogenic lipocalin 24p3 antibody
    • p25 antibody
    • Siderocalin antibody
    • siderocalin LCN2 antibody
    • SV40 induced 24P3 protein antibody
    • Uterocalin antibody
    see all

画像

  • Anti-Lipocalin-2 / NGAL antibody (ab41105) at 1 µg/ml + Human colon tissue lysate - total protein (ab30051) at 30 µg

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 23 kDa
    Observed band size: 21 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 25 kDa, 70 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 12 minutes
  • ICC/IF image of ab41105 stained human Hek293 cells. The cells were methanol fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab41105, 5µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

  • ab41105 staining Lipocalin-2 / NGAL in human lung tissue by Immunohistochemistry (Formalin/PFA-fixed paraffin embedded sections). Tissue underwent fixation in formaldehyde, heat mediated antigen retrieval in Citrate buffer pH6.0 and blocking for 15 minutes at 20°C (5 minutes peroxidase block and 10 minutes protein blocks). The primary antibody was diluted 1/250 or 1/1000 and incubated  with sample for 45 minutes at 20°C. A HRP-conjugated goat polyclonal to rabbit IgG was used undiluted as the secondary.   

    See Abreview

  • ab41105 staining Lipocalin-2 / NGAL in rat lung tissue section by Immunohistochemistry (Formalin/PFA-fixed paraffin embedded sections). Tissue underwent fixation in formaldehyde, heat mediated antigen retrieval in Citrate buffer pH6.0 and blocking for 15 minutes at 20°C (5 minutes peroxidase block and 10 minutes protein blocks). The primary antibody was diluted 1/250 or 1/1000 and incubated  with sample for 45 minutes at 20°C. A HRP-conjugated goat polyclonal to rabbit IgG was used undiluted as the secondary. 

    See Abreview

参考文献

This product has been referenced in:
  • De La Chesnaye E  et al. Expression profiling of lipocalin-2 and 24p3 receptor in murine gonads at different developmental stages. Exp Ther Med 16:213-221 (2018). Read more (PubMed: 29896242) »
  • Cortés-Araya Y  et al. Comparison of Antibacterial and Immunological Properties of Mesenchymal Stem/Stromal Cells from Equine Bone Marrow, Endometrium, and Adipose Tissue. Stem Cells Dev 27:1518-1525 (2018). Read more (PubMed: 30044182) »
See all 9 Publications for this product

レビューと Q&A

1-10 of 11 Abreviews or Q&A

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Tissue sections (heart)
Permeabilization
Yes - 0.3% Triton X
Specification
heart
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 22°C
Fixative
Acetone

Abcam user community

Verified customer

投稿 May 09 2017

Abcam has not validated the combination of species/application used in this Abreview.
Application
Immunohistochemistry (Frozen sections)
Sample
Rat Tissue sections (liver)
Permeabilization
Yes - 0.1% Triton X
Specification
liver
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 22°C
Fixative
Acetone

Abcam user community

Verified customer

投稿 May 08 2017

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (human sputum)
Loading amount
20 µg
Specification
human sputum
Gel Running Conditions
Reduced Denaturing (15%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C

Mr. Taehyeong lee

Verified customer

投稿 Nov 16 2012

Answer

Thank you for contacting us. I am sorry that these antibodies are not giving any signal in IHC.

Have you been able to use the secondary antibody successfully with any other primaries? If not, I would recommend spotting the secondary and DAB reagents together on parafilm to see if you get any color change. If not, it would indicate that either the HRP on the secondary is inactive, or the DAB is no longer active. This seems to be the most likely situation, because using both the primary and secondary antibodies undiluted should at least show background signal.

If you get color development from the DAB, I would recommend trying a longer antigen retrieval (10-15 minutes with the pressure cooker). As a rule of thumb, if you do not observe any signal, the antigen retrieval time may be too short. If you observe high background, it may be too long. What buffer are you using for antigen retrieval? This antibody was validated using citrate buffer at pH 6.

I hope this helps to improve your results, if not, please let me know your original order number and I will be happy to help you further.

Read More

Answer

Thank you for taking time to answer my questions. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed this case, I would like to offer some suggestions to help optimize the results from ab41105.

1. Load 50 ug of protein rather than 30 ug. Previous customers have used this amount and have got excellent results.

2. Increase the boiling time to 10 minutes rather than 3 minutes to disrupt any multimers which may form.

These are the main suggestions but you could also try increasing the concentration of the blocking agent to 5% BSA ( which most customers have used) or increasing the antibody concentration to 1:300.

Should the suggestions not improve the results, please do let me know.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Read More

Answer

Thank you for taking time to send us your protocol. I am sorry to hear that this antibody is not providing satisfactory results.

The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

Having reviewed your protocol, for me to be able to give you the best possible technical help, could you confirm the following details:

1. Sample preparation:
Type of sample (whole cell lysates, fraction, recombinant protein…):
Lysis buffer
Protease inhibitors:
Phosphatase inhibitors
Reducing agent
Boiling for ≥5 min? yes/no
Protein loaded ug/lane or cells/lane
Positive control
Negative control

2. Incubation time and temperature with blocking agent?

3. Incubation time and temperature with primary antibody?

4. Incubation time and temperature with secondary antibody?

I also noticed that 2 your samples were mouse intestine and pig intestine. Unfortunately this antibody has not been tested in these species and therefore are not guaranteed to work. I have done a sequence comparison between human Lipocalin 2 and mouse and pig Lipocalin 2. These only show 60.6% and 70.7% sequence similarity respectively and therefore are unlikely to be detected by this antibody hence why no band is seen for these 2 samples.

For your CACO-2 and HUVEC cells, an band should have been detected but on your blot it is very faint. Once you have sent me the additional information asked, I will see whether I can make any suggestions to improve your results.

In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

I hope this information is helpful, and I thank you for your cooperation.

Read More

Answer

Thank you for providing that information. This will be very useful in investigating your complaint further.


If you could provide me with the order number on which the antibody was ordered (or the approximate delivery date and delivery address) I can organise for a full refund to be paid.

Read More
Application
Western blot
Sample
Rat Tissue lysate - whole (Lung)
Loading amount
50 µg
Specification
Lung
Gel Running Conditions
Reduced Denaturing (12)
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C

Abcam user community

Verified customer

投稿 Aug 17 2011

Application
Western blot
Sample
Rat Cell lysate - whole cell (Hepatocytes)
Loading amount
50 µg
Specification
Hepatocytes
Gel Running Conditions
Reduced Denaturing (12%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Dr. Shakil Ahmad

Verified customer

投稿 Aug 09 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (lung)
Specification
lung
Fixative
Formaldehyde
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Citrate buffer pH6.0
Permeabilization
No
Blocking step
5 minutes of peroxidase block then 10 minutes of protein block. These are ready-to-use reagents purchased from Dako as blocking agent for 15 minute(s) · Concentration: 100% · Temperature: 20°C

Mr. Antibody Solutions

Verified customer

投稿 Oct 28 2008

1-10 of 11 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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