Overview

  • Product name
    Anti-Proteasome 20S LMP7 antibody
    See all Proteasome 20S LMP7 primary antibodies
  • Description
    Rabbit polyclonal to Proteasome 20S LMP7
  • Host species
    Rabbit
  • Specificity
    Detects proteasome 20S LMP7.
  • Tested applications
    Suitable for: WB, IHC-P, ICC/IF, IHC-Fr, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Sheep, Cow
  • Immunogen

    Synthetic peptide corresponding to Human Proteasome 20S LMP7 aa 259-274.
    Sequence:

    VESTDVSDLLHQYREA


    Database link: P28062
    (Peptide available as ab4945)

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage buffer
    Constituents: 0.1% BSA, 99% PBS
  • Concentration information loading...
  • Purity
    Immunogen affinity purified
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab3329 in the following tested applications.

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

Application Abreviews Notes
WB Use a concentration of 1 - 3 µg/ml. Detects a band of approximately 20 kDa.Can be blocked with Human Proteasome 20S LMP7 peptide (ab4945).
IHC-P Use a concentration of 1 µg/ml.
ICC/IF Use a concentration of 5 µg/ml.
IHC-Fr 1/500. PubMed: 17008387
Flow Cyt Use 3-5µg for 106 cells.

Target

  • Function
    The proteasome is a multicatalytic proteinase complex which is characterized by its ability to cleave peptides with Arg, Phe, Tyr, Leu, and Glu adjacent to the leaving group at neutral or slightly basic pH. The proteasome has an ATP-dependent proteolytic activity. This subunit is involved in antigen processing to generate class I binding peptides. Replacement of PSMB5 by PSMB8 increases the capacity of the immunoproteasome to cleave model peptides after hydrophobic and basic residues. Acts as a major component of interferon gamma-induced sensitivity. Plays a key role in apoptosis via the degradation of the apoptotic inhibitor MCL1. May be involved in the inflammatory response pathway. In cancer cells, substitution of isoform 1 (E2) by isoform 2 (E1) results in immunoproteasome deficiency.
  • Involvement in disease
    Defects in PSMB8 are the cause of JMP syndrome (JMPS) [MIM:613732]; also called joint contractures muscular atrophy microcytic anemia and panniculitis-induced lipodystrophy. JBTS1 is an autoinflammatory disorder characterized by childhood onset of joint stiffness and severe contractures of the hands and feet, erythematous skin lesions with subsequent development of severe lipodystrophy, and laboratory evidence of immune dysregulation. Accompanying features include muscle weakness and atrophy, hepatosplenomegaly, and microcytic anemia.
  • Sequence similarities
    Belongs to the peptidase T1B family.
  • Developmental stage
    Highly expressed in immature dendritic cells (at protein level).
  • Post-translational
    modifications
    Autocleaved. The resulting N-terminal Thr residue of the mature subunit is responsible for the nucleophile proteolytic activity.
  • Cellular localization
    Cytoplasm. Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • ALDD antibody
    • D6S216 antibody
    • D6S216E antibody
    • Large multifunctional peptidase 7 antibody
    • Large multifunctional protease 7 antibody
    • LMP 7 antibody
    • LMP7 antibody
    • Low molecular mass protein 7 antibody
    • Low molecular weight protein 7 antibody
    • Macropain subunit C13 antibody
    • MGC1491 antibody
    • Multicatalytic endopeptidase complex subunit C13 antibody
    • NKJO antibody
    • OTTHUMP00000062981 antibody
    • Protease component C13 antibody
    • Proteasome (prosome macropain) subunit beta type 8 antibody
    • Proteasome (prosome, macropain) subunit, beta type, 8 (large multifunctional peptidase 7) antibody
    • Proteasome beta 8 subunit antibody
    • Proteasome catalytic subunit 3i antibody
    • Proteasome component C13 antibody
    • Proteasome related gene 7 antibody
    • Proteasome subunit beta 5i antibody
    • Proteasome subunit beta 8 antibody
    • Proteasome subunit beta type 8 antibody
    • Proteasome subunit beta type antibody
    • Proteasome subunit beta type-8 antibody
    • Proteasome subunit beta-5i antibody
    • Proteasome subunit Y2 antibody
    • PSB8_HUMAN antibody
    • PSMB 8 antibody
    • PSMB5i antibody
    • PSMB8 antibody
    • Really interesting new gene 10 protein antibody
    • RING 10 antibody
    • RING10 antibody
    • Y2 antibody
    see all

Images

  • All lanes : Anti-Proteasome 20S LMP7 antibody (ab3329) at 2 µg/ml

    Lane 1 : HeLa whole cell extracts
    Lane 2 : HeLa treated with IFN gamma (100ng/ml IFN gamma for 72h) whole cell extracts
    Lane 3 : U-937 whole cell extracts
    Lane 4 : Raji whole cell extracts
    Lane 5 : Ramos whole cell extracts

    Lysates/proteins at 30 µg per lane.

    Secondary
    All lanes : Goat anti-Rabbit IgG (H+L) HRP conjugate at 1/2500 dilution

    Observed band size: 20 kDa
    why is the actual band size different from the predicted?



    Detected by chemiluminescence.

  • Immunocytochemistry/ Immunofluorescence analysis of HeLa cells labeling Proteasome 20S LMP7 with ab3329 at 5µg/ml. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 in TBS for 10 minutes, and blocked with 3% Blocker BSA in PBS for 15 minutes at room temperature. Cells were stained with or without Anti-Proteasome 20S LMP7 antibody (ab3329), at a concentration of 5µg/ml for 1 hour at room temperature, and then incubated with a Alexa Fluor® 488 goat anti-rabbit IgG secondary antibody at a dilution of 1/1000 for 1 hour s at room temperature (both panels, green). Nuclei (both panels, blue) were stained with Hoechst 33342 dye.

  • Ab3329 staining Human normal skin. Staining is localized to cytoplasmic and nuclear compartments.
    Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control.
    Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer citrate pH 6.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS), then incubated with primary antibody for 20 minutes, and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required
  • Flow Cytometry analysis of SH-SY5Y cells labeling Proteasome 20S LMP7 with ab3329. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with Anti-Proteasome 20S LMP7 antibody (ab3329) (red histogram) or with rabbit isotype control (pink histogram) at 3-5 ug/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Goat Anti-Rabbit Secondary Antibody at a dilution of 1/400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

  • Western blot of proteasome 20S LMP7 from HeLa cell extract using ab3329.
  • ICC/IF image of ab3329 stained MCF7 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab3329, 5µg/ml) overnight at +4°C. 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) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:
  • Ramos de Carvalho JE  et al. Involvement of the ubiquitin-proteasome system in the expression of extracellular matrix genes in retinal pigment epithelial cells. Biochem Biophys Rep 13:83-92 (2018). Read more (PubMed: 29387813) »
  • Ugras S  et al. Induction of the Immunoproteasome Subunit Lmp7 Links Proteostasis and Immunity in a-Synuclein Aggregation Disorders. EBioMedicine 31:307-319 (2018). WB, IHC-P ; Mouse, Human . Read more (PubMed: 29759483) »
See all 24 Publications for this product

Customer reviews and Q&As

1-10 of 14 Abreviews or Q&A

Application
Western blot
Sample
Human Cell lysate - whole cell (HTB-16 cells)
Gel Running Conditions
Reduced Denaturing (12%)
Loading amount
10 µg
Treatment
1ug INFgamma for 12hrs
Specification
HTB-16 cells
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 24°C

Abcam user community

Verified customer

Submitted Oct 08 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Tissue lysate - whole (Skeletal muscle)
Gel Running Conditions
Reduced Denaturing (12%)
Loading amount
20 µg
Specification
Skeletal muscle
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Feb 29 2016

Application
Western blot
Sample
Mouse Tissue lysate - whole (Skeletal muscle)
Gel Running Conditions
Reduced Denaturing (12%)
Loading amount
20 µg
Specification
Skeletal muscle
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Feb 29 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Tissue lysate - whole (Skeletal muscle)
Loading amount
30 µg
Specification
Skeletal muscle
Gel Running Conditions
Reduced Denaturing (15% gel)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C

Dr. G.K Sakellariou

Verified customer

Submitted May 15 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Recombinant protein (Recombinant LMP7-GST)
Loading amount
0.025 µg
Specification
Recombinant LMP7-GST
Gel Running Conditions
Reduced Denaturing (4-12% bis/tris)
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Abcam user community

Verified customer

Submitted May 31 2012

Answer

Thank you for sending me this image. This data will be used to keep records of how our products are performing and for internal invetigation if needed. I noted that all tissue lysates that you have used have given the same non-specific signal. However I was wondering if you had tried a positive control lysate with your experiments. I ask this because according to SwissProt, in mouse tissue,this protreasome is expressed in spleen, thymus, lung, liver, heart and, at a very low level, in kidney.However it is not expressed in brain nor testis. My concern is that whilethe antibody may be recognizing non-specifically it may still be performing as there is not expression in the brain. This information is in the general annotations on this page: http://www.uniprot.org/uniprot/P28063 I would highly recommend using a control tissue lysate such as ab7937 Spleen (Mouse) Whole Cell Lysate - normal tissue ( https://www.abcam.com/Spleen-Mouse-Whole-Cell-Lysate-normal-tissue-ab7937.html). If you have any questions please do not hesitate to contact me.

Read More

Answer

Thank you for taking time to send us your protocols. 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 ab38543. Spotty/sandy looking blots are often the result of aggregates in the block or antibody. I would recommend when preparing the milk in TBST that you stir it with a stir bar for quite some time. This will help dissolve particulate matter that can stick to your blot. Filtering the milk will also remove any milk that has not dissolved. I would also suggest that you centrifuge the antibody before use and use the supernatant from the top should aggregates appear. You may use the supernatant at recommended dilutions without a reduction in quality. I would also recommend using a lower concentration for your secondary antibody. Some people have noted that rabbit secondaries used at high concentration may cause issues like this. To increase target signal, I would suggest using a higher concentration of ant- SMURF 2 antibody. This product has been used successfully at concentrations as high as 1:50. 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. In regards to ab3329. I've seen that others have posted abreviews which have shown a higher non-specific band at about 66 kDa. Is this about the range of the higher band that you have reported? Do you have an image that you might send us? Would it also be possible to send me the original order number or purchase order for these products. If we need to replace them having that information will expedite the process. I hope this information is helpful, and I thank you for your cooperation.

Read More

Answer

Thank you for contacting us. I would like to ask you if you might send me the protocols used with these productsso that we can gather further information regarding the samples tested and the protocol used. Once we receive your response, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody. For our records could you please include the following details in your protocols for each antibody? What tissue type was used? How was the tissue prepared, including lysis buffer and conditions? Were the samples denatured? How much total protein was loaded per well? What were the running and transfer conditions and buffers? Did you use a loading control? Did you usethe recommended positive controls? What were the results of those controls? What blocking reagents did you use? You have mentioned that milk and BSA were used for ab3329, what were the concentrations of these in your block? What incubation times and temperatures did you use? What concentrations did you use the primary antibody concentration at? What incubation times, temperatures and reagents did you use? What was the secondary antibody? What were the concentrations, incubation times, temperatures and reagents used with this? Were different conditions tried, including different antibody concentrations and incubation times? What wash steps did you use? Which detection system did you use? I look forward to hearing form you soon.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (dendritic cell)
Loading amount
1e+006 cells
Specification
dendritic cell
Gel Running Conditions
Non-reduced Denaturing
Blocking step
BSA as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Jan 05 2010

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Purified protein (heart and spleen)
Loading amount
1 µg
Specification
heart and spleen
Gel Running Conditions
Reduced Denaturing (12,5%)
Blocking step
Milk as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Sep 03 2008

1-10 of 14 Abreviews or Q&A

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