Overview

  • Product name
    Anti-Proteasome 19S S5A / ASF antibody [AH1.1]
    See all Proteasome 19S S5A / ASF primary antibodies
  • Description
    Mouse monoclonal [AH1.1] to Proteasome 19S S5A / ASF
  • Host species
    Mouse
  • Tested applications
    Suitable for: Flow Cyt, ICC/IF, IHC-Fr, IP, WBmore details
  • Species reactivity
    Reacts with: Human, Pig, Xenopus laevis
    Does not react with: Mouse
  • Immunogen

    Other Immunogen Type corresponding to Proteasome 19S S5A/ ASF.

  • General notes

    Previously labelled as Proteasome 19S S5A. 

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
    Constituent: PBS
  • Concentration information loading...
  • Purity
    Protein A/G purified
  • Clonality
    Monoclonal
  • Clone number
    AH1.1
  • Myeloma
    Sp2/0-Ag14
  • Isotype
    IgG1
  • Light chain type
    unknown
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab20239 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
Flow Cyt Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

ICC/IF 1/200.
IHC-Fr 1/100. Fix with acetone.
IP Use a concentration of 5 µg/ml.
WB Use at an assay dependent concentration. Predicted molecular weight: 41 kDa.

Target

  • Function
    Binds and presumably selects ubiquitin-conjugates for destruction. Displays selectivity for longer polyubiquitin chains. Modulates intestinal fluid secretion.
  • Sequence similarities
    Belongs to the proteasome subunit S5A family.
    Contains 2 UIM (ubiquitin-interacting motif) repeats.
    Contains 1 VWFA domain.
  • Domain
    The 2 UIM motifs are involved in the binding to a multi-ubiquitin chain in a cooperative way.
  • Post-translational
    modifications
    Phosphorylated upon DNA damage, probably by ATM or ATR.
  • Information by UniProt
  • Database links
  • Alternative names
    • 26S protease subunit S5a antibody
    • 26S proteasome non ATPase regulatory subunit 4 antibody
    • 26S proteasome non-ATPase regulatory subunit 4 antibody
    • 26S proteasome regulatory subunit rpn10 antibody
    • 26S proteasome regulatory subunit S5A antibody
    • AF 1 antibody
    • AF antibody
    • AF1 antibody
    • Angiocidin antibody
    • Antisecretory factor 1 antibody
    • ASF antibody
    • DS5a antibody
    • MCB 1 antibody
    • MCB1 antibody
    • Multiubiquitin chain binding protein antibody
    • Multiubiquitin chain-binding protein antibody
    • OTTHUMP00000059963 antibody
    • Prosome macropain antibody
    • Proteasome (prosome macropain) 26S subunit non ATPase 4 antibody
    • Proteasome 19S S5A antibody
    • Proteasome 26S non ATPase subunit 4 antibody
    • Proteasome 26S subunit non ATPase 4 antibody
    • PSMD 4 antibody
    • Psmd4 antibody
    • PSMD4_HUMAN antibody
    • pUB R5 antibody
    • pUBR5 antibody
    • Rpn 10 antibody
    • Rpn10 antibody
    • RPN10 homolog antibody
    • S5A antibody
    • S5a/antisecretory factor protein antibody
    see all

Images

  • Proteasome 19S S5A / ASF was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Mouse monoclonal to Proteasome 19S S5A / ASF and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

    The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab20239.

    Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/20,000 dilution.

    Band: 41kDa; Proteasome 19S S5A / ASF

  • ab20239 (1/200) staining Proteasome 19S S5A / ASF in Human retinal pigment epithelial (RPE) cells (green). Cells were fixed in Paraformaldehyde, permeabilised with 0.5% Triton X100/PBS and counterstained with DAPI in order to highlight the nucleus (blue). Please refer to Abreview for further details.

    See Abreview

  • Overlay histogram showing HeLa cells stained with ab20239 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab20239, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed.

References

This product has been referenced in:
  • Liu YP  et al. Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators. J Clin Invest 124:2059-70 (2014). WB . Read more (PubMed: 24691443) »
  • Kley RA  et al. Pathophysiology of protein aggregation and extended phenotyping in filaminopathy. Brain 135:2642-60 (2012). Read more (PubMed: 22961544) »
See all 5 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Answer

Thank you for contacting Abcam.

I have heard back from the lab and they have told me that with regard to your queries:

1) I believe that the antibody does cross react with mouse but unfortunately do not have any confirmed evidence that I can send for this.

2) Again we do not have any actual evidence for this but as the immunogen was the specific protein subunit we would very much expect the antibody to have been generated against the peptide.

3) We are not be able to provide the peptide for ab20239.

As we are unsure if the antibody would react with mouse (we had some previous indication that it would not), then the next time you can take advantage of our testing discount program:

https://www.abcam.com/index.html?pageconfig=resource&rid=11998&viapagetrap=collaborationdiscount

Please let me know if there is anything else I can help you with.

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Answer

I am very pleased to hear you would like to accept our offer and test these products in zebrafish. Each code will give you 1 free PRIMARY ANTIBODY before the expiration date. To redeem this offer, please submit an Abreview for zebrafish and include this code in the “Additional Comments” section so we know the Abreview is for this promotion. For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews. Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code. Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research. The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

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Answer

Thank you for contacting Abcam. I am sorry about the delay in answering your questions as I have been waiting for the lab to respond.

According to the information that I have been able to obtain, ab20239 will not react with mouse S5A. We also can not currently offer the protein sample used to create this antibody.

GAPDH has not been tested in zebrafish samples. However there is an 86% homology between the immunogen and zebrafish. If you would be interested in also participating in the Abreview program for that product I could create a code for Ab9484 as well. Our Mouse monoclonal [6C5] to GAPDH (ab8245) has been tested in zebrafish and is guaranteed under our Abpromise.

If you would like the 100% Abreview code for ab9484 as well as the code for ab20239 please let me know. I look forward to hearing from you.

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Answer

Thank you for contacting us.


This products immunogen is from the full S5A protein. We do not presently know the epitope that the antibody reacts against. I did run alignments for you against D.rerio and found84% identity between the human and D.rerio psmd4a sequence, 91% positives and that the D.rerio psmd4b sequence is 80% identity and 90% positive.

Although we have not tested this product in zebrafish, these numbers make it likely that this could work in that species.Therefore, I can offer a discount off a future purchase if you buy ab20239 now, test it in zebrafish and submit feedback to us in the form of an Abreview. It doesn’t matter whether the Abreview is positive or negative, we would just really like to receive your feedback. The discount would be to the value of 1 free PRIMARY ANTIBODY.If you are interested in this offer, I can send you more information.

Please let me know if you have any questions about this offer and I would be happy to help you further.

The Terms and Conditions of this offer can be found at: www.abcam.com/collaborationdiscount

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Answer

Thank you for contacting us. That is correct, you should receive 91 ul.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (HEK293T)
Gel Running Conditions
Reduced Denaturing (4-12% invitrogen)
Loading amount
5 µg
Treatment
siRNA
Specification
HEK293T
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C

Dr. Tibor Pankotai

Verified customer

Submitted Sep 16 2011

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (RPE-1)
Permeabilization
Yes - 0.5% Triton X100 in PBS
Specification
RPE-1
Fixative
Paraformaldehyde

Dr. Kirk Mcmanus

Verified customer

Submitted Aug 14 2007

Question

BATCH NUMBER 147111 ORDER NUMBER 119732 DESCRIPTION OF THE PROBLEM ECL: no signal at all. Odyssey: weak signal + non-specific bands. Some background on the membrane detected, too. SAMPLE Mouse, NSC34 neuroblastoma cell extract. PRIMARY ANTIBODY In all cases, ABCAM ab20239 anti-mouse S5a, monoclonal made in mouse. For ECL: in 5% non-fat dry milk in PBST OR TBST. Dilution: 1:1000. Incubation: overnight, +4 C. For Odyssey, in Odyssey Blocking Buffer (undiluted) with 0.1% Tween-20. Dilution: 1:1,000 OR 1:3,000. Incubation: overnight, +4 C. Wash: 4X5 minutes. ECL: PBST or TBST; Odyssey: PBST. DETECTION METHOD ECL: Pierce Supersignal West Pico Chemiluminescent Substrate. Odyssey: Li-Cor scanner. POSITIVE AND NEGATIVE CONTROLS USED No (I don't have purified S5a protein, nor a surely-working cellular extract). ANTIBODY STORAGE CONDITIONS The antibody arrived yesterday. Since then, it was stored at +4 C. SAMPLE PREPARATION The sample was prepared in 1X RIPA buffer (Upstate) supplemented with Sigma P-8340 protease inhibitors (1:300). The cell pellet was homogenized into the buffer by sucking up-down 20 times in a syringe with a 26G needle. The lysate was kept on ice for 20 minutes, then centrifuged at 1,000 G at 4C for 10 minutes. The supernatant was used. Sample: heated (95 C, 5 minutes) in SDS loading buffer before loading on the gel. AMOUNT OF PROTEIN LOADED 50 micrograms total protein (as determined by the Bio-Rad Protein Assay Reagent, it is kind of a Bradford reagent). ELECTROPHORESIS/GEL CONDITIONS 10% acrylamide, SDS-PAGE, non-reducing gel (but the sample loading buffer was reducing, it contained DTT). TRANSFER AND BLOCKING CONDITIONS Transfer: to nitrocellulose membrane, in TRIS-glycine buffer (3g TRIS, 14.4g glycine per liter) with 20% methanol; 70V, 90 minutes, kept on ice during transfer. Blocking: in all cases, 1 hour at room temperature. For ECL, 5% non-fat dry milk in 1XPBS/0.1% Tween-20 (PBST) OR 5% non-fat dry milk in 1XTBS (100mM TRIS-HCl, pH 7.5; 0.9% NaCl)/0.1% Tween-20 (TBST). For Odyssey, Odyssey Blocking Buffer diluted 1:1 with 1XPBS. SECONDARY ANTIBODY For ECL: Sigma A5278 anti-mouse IgG (whole molecule), HRP-conjugated. Diluted in 5% non-fat dry milk in PBST OR TBST. Dilution: 1:10,000. Incubation: 1.5 hours, room temperature. For Odyssey: goat anti-mouse, IRDye 800 conjugate (Li-Cor), 1:10,000 in Odyssey Blocking Buffer with 0.1% Tween-20. 1 hour, room temperature. Wash: 4X5 minutes. ECL: PBST or TBST; Odyssey: PBST. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No ADDITIONAL NOTES We have recently purchased your ab20239 anti-S5a antibody. I tried it on an NSC34 cellular extract prepared in RIPA buffer. I have run the sample on four lanes with 50 micrograms total protein sample per each lane on a 10% gel alongside a marker, transferred the proteins to a nitrocellulose membrane, cut the membrane in four pieces, and tried two Western methods: ECL (using PBST or TBST) and the Li-Cor Odyssey System (based on infrared fluorescence) using two primary antibody dilutions for Odyssey: 1:1,000 and 1:3,000. With ECL, I did not get ANY signal. With Odyssey, I got bands, but the signal is rather weak, and additional bands, both at higher- and lower-than-expected MW-s appeared. I attach the Odyssey results (S5a.jpg). In both cases, the marker is the Bio-Rad Low-Range Prestained Marker. On the left, the sample was probed with the ab20239 antibody at 1:3,000 dilution; on the right, at 1:1,000 dilution. I think that 2 or 3 of the bands in the middle of the lanes might be real; there are S5a splice variants between 41 and 28 kDa, but at least the 41 kDa form was reported to have an anomalous running at about 60 kDa. I suppose that Abcam has tested this antibody against mouse cellular extracts. I would really appreciate if you could supply me with some recommendations on how I could improve my results.

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Answer

I have just received feedback from the source of ab20239 regarding your problem and have been informed that the information we received was wrong and that the antibody did not work very well in mouse samples, but worked better in human samples. My sincere apologies for this error, I would like to offer you a refund or credit note on your order of this antibody, if you could let me know which you would prefer. I believe your order is number 119732 for the University of Kentucky. I have immediately corrected the datasheet and once again hope you can accept my apologies for this unfortunate mistake. I look forward to hearing from you regarding your preference of a refund or credit note,

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Question

BATCH NUMBER 147111 ORDER NUMBER 119732 DESCRIPTION OF THE PROBLEM ECL: no signal at all. Odyssey: weak signal + non-specific bands. Some background on the membrane detected, too. SAMPLE Mouse, NSC34 neuroblastoma cell extract. PRIMARY ANTIBODY In all cases, ABCAM ab20239 anti-mouse S5a, monoclonal made in mouse. For ECL: in 5% non-fat dry milk in PBST OR TBST. Dilution: 1:1000. Incubation: overnight, +4 C. For Odyssey, in Odyssey Blocking Buffer (undiluted) with 0.1% Tween-20. Dilution: 1:1,000 OR 1:3,000. Incubation: overnight, +4 C. Wash: 4X5 minutes. ECL: PBST or TBST; Odyssey: PBST. DETECTION METHOD ECL: Pierce Supersignal West Pico Chemiluminescent Substrate. Odyssey: Li-Cor scanner. POSITIVE AND NEGATIVE CONTROLS USED No (I don't have purified S5a protein, nor a surely-working cellular extract). ANTIBODY STORAGE CONDITIONS The antibody arrived yesterday. Since then, it was stored at +4 C. SAMPLE PREPARATION The sample was prepared in 1X RIPA buffer (Upstate) supplemented with Sigma P-8340 protease inhibitors (1:300). The cell pellet was homogenized into the buffer by sucking up-down 20 times in a syringe with a 26G needle. The lysate was kept on ice for 20 minutes, then centrifuged at 1,000 G at 4C for 10 minutes. The supernatant was used. Sample: heated (95 C, 5 minutes) in SDS loading buffer before loading on the gel. AMOUNT OF PROTEIN LOADED 50 micrograms total protein (as determined by the Bio-Rad Protein Assay Reagent, it is kind of a Bradford reagent). ELECTROPHORESIS/GEL CONDITIONS 10% acrylamide, SDS-PAGE, non-reducing gel (but the sample loading buffer was reducing, it contained DTT). TRANSFER AND BLOCKING CONDITIONS Transfer: to nitrocellulose membrane, in TRIS-glycine buffer (3g TRIS, 14.4g glycine per liter) with 20% methanol; 70V, 90 minutes, kept on ice during transfer. Blocking: in all cases, 1 hour at room temperature. For ECL, 5% non-fat dry milk in 1XPBS/0.1% Tween-20 (PBST) OR 5% non-fat dry milk in 1XTBS (100mM TRIS-HCl, pH 7.5; 0.9% NaCl)/0.1% Tween-20 (TBST). For Odyssey, Odyssey Blocking Buffer diluted 1:1 with 1XPBS. SECONDARY ANTIBODY For ECL: Sigma A5278 anti-mouse IgG (whole molecule), HRP-conjugated. Diluted in 5% non-fat dry milk in PBST OR TBST. Dilution: 1:10,000. Incubation: 1.5 hours, room temperature. For Odyssey: goat anti-mouse, IRDye 800 conjugate (Li-Cor), 1:10,000 in Odyssey Blocking Buffer with 0.1% Tween-20. 1 hour, room temperature. Wash: 4X5 minutes. ECL: PBST or TBST; Odyssey: PBST. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No ADDITIONAL NOTES We have recently purchased your ab20239 anti-S5a antibody. I tried it on an NSC34 cellular extract prepared in RIPA buffer. I have run the sample on four lanes with 50 micrograms total protein sample per each lane on a 10% gel alongside a marker, transferred the proteins to a nitrocellulose membrane, cut the membrane in four pieces, and tried two Western methods: ECL (using PBST or TBST) and the Li-Cor Odyssey System (based on infrared fluorescence) using two primary antibody dilutions for Odyssey: 1:1,000 and 1:3,000. With ECL, I did not get ANY signal. With Odyssey, I got bands, but the signal is rather weak, and additional bands, both at higher- and lower-than-expected MW-s appeared. I attach the Odyssey results (S5a.jpg). In both cases, the marker is the Bio-Rad Low-Range Prestained Marker. On the left, the sample was probed with the ab20239 antibody at 1:3,000 dilution; on the right, at 1:1,000 dilution. I think that 2 or 3 of the bands in the middle of the lanes might be real; there are S5a splice variants between 41 and 28 kDa, but at least the 41 kDa form was reported to have an anomalous running at about 60 kDa. I suppose that Abcam has tested this antibody against mouse cellular extracts. I would really appreciate if you could supply me with some recommendations on how I could improve my results.

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Answer

I'm sorry to hear you are having a problem with ab20239. According to your order shipping details the antibody was shipped to you very rapidly with no delay, and we have not received any other complaints about this antibody. I am currently in touch with the source of this antibody to find out the recommended dilution in WB, recommended positive control and to clarify what type of blocking was used, as it is likely that the multiple bands is due to the blocking agent. Indeed your protocol looks very good and may simply need the following optimisation steps: -a 1hr blocking in 5%BSA in TBST -a 1:300-1:500 dilution of the antibody (in TBST, no blocking agent) (overnight incubation) -incubation of the secondary antibody in TBST only. In our laboratory we also use both ECl+ and the Odyssey but I would expect the antibody to already work with the less sensitive system. I will let you know as soon as I receive information from the supplier of the antibody to confirm the dilution they had used and positive control, I apologise for the delay and hope the recommendations above will already help,

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