Overview

  • Product name
    Anti-Proteasome 20S alpha + beta antibody
  • Description
    Rabbit polyclonal to Proteasome 20S alpha + beta
  • Host species
    Rabbit
  • Tested applications
    Suitable for: ICC, IHC-Fr, WB, IP, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human, Saccharomyces cerevisiae
  • Immunogen

    Tissue/ cell preparation: Proteasomal preparation isolated from human red blood cells.

  • Positive control
    • Human and yeast 20S proteasome and tissue/cell preparations including human placental proteasome preparation, a HeLa cell lysate, RSV 3T3 mouse fibroblast cell lysate, and yeast whole cell extract.
  • General notes
    Dilute to working concentration in PBS pH 7.2- 7.4, and store at 2 to 4°C (do not freeze). Use diluted antibody within 1 month.

Properties

Applications

Our Abpromise guarantee covers the use of ab22673 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
ICC Use at an assay dependent concentration. PubMed: 23840376
IHC-Fr Use at an assay dependent concentration.
WB 1/1000 - 1/10000. Predicted molecular weight: 25 - 30 kDa.
IP Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration. See Abreview.

Target

Images

  • ab22673 staining human Hela cells by ICC/IF. Cells were formaldehyde fixed, permeabilized in 0.1% Triton and blocked in 3% BSA for 15 minutes at room temperature. The primary antibody was diluted 1/2000 (3% BSA) and incubated with sample for 1 hour. An Alexa Fluor® 488 conjugated goat polyclonal to rabbit, diluted 1/1000 was used as secondary.

    See Abreview

  • All lanes : Anti-Proteasome 20S alpha + beta antibody (ab22673) at 1/2000 dilution

    All lanes : Whole tissue lysate prepared from rat prefrontal cortex

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat anti-rabbit IgG-HRP at 1/5000 dilution

    Developed using the ECL technique.

    Predicted band size: 25 - 30 kDa


    Exposure time: 5 minutes


    Blocked with 5% milk for 30 minutes at 27°C.

    See Abreview

  • Immunofluorescence analysis of HeLa cells, staining Proteasome 20S alpha + beta with ab22673.

    Cells were fixed with formaldehyde and permeabilized using 0.1% Triton X-100. Samples were incubated with primary antibody (1/1000) fr 1 hour at room temperature. An AlexaFluor®555-conjugated donkey anti-rabbit IgG was used as the secondary antibody.

References

This product has been referenced in:
  • Steen KA  et al. FABP4/aP2 Regulates Macrophage Redox Signaling and Inflammasome Activation via Control of UCP2. Mol Cell Biol 37:N/A (2017). WB ; Mouse . Read more (PubMed: 27795298) »
  • Zhao Y  et al. Essential role of proteasomes in maintaining self-renewal in neural progenitor cells. Sci Rep 6:19752 (2016). WB, ICC/IF ; Mouse . Read more (PubMed: 26804982) »
See all 13 Publications for this product

Customer reviews and Q&As

1-10 of 14 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Sample
Human Cell (HS 5)
Specification
HS 5
Permeabilization
Yes - Triton X 100
Fixative
Formaldehyde

Mr. Kannan Govindaraj

Verified customer

Submitted Aug 02 2013

Answer

Thank you for contacting us yesterday.

I am sorry but as you are aware we do not currently have any of the Anti-Proteasome 20S alpha + beta antibody (ab22673). As discussed over the phone, I have had a look for antibodies which may serve as suitable alternatives for you. Please have a look at the following antibodies to see if they may be suitable for your experiments:

Rabbit polyclonal to Proteasome 20S LMP7 ab3329

Rabbit polyclonal to Proteasome 20S C2 ab3325

Rabbit polyclonal to Proteasome 20S LMP2 ab42987

Mouse monoclonal [MCP231] to Proteasome 20S alpha 1+2+3+5+6+7 ab22674

I hope this information has been of help. If you have any further questions, please do not hesitate to contact us again.

Read More

Answer

Thank you for your reply.

I can change the antibody on your current order from ab22673 for ab109525 if you would like. As this is in stock, if you let me know you would like for me to do this, it can be with you by tomorrow.

The antibody has been used both in WB and IF and should therefore be suitable for your experiments. Please be aware that this antibody is raised against the Proteasome 20S alpha 2 and will detect this protein. It has been shown to work with mouse, rat and human samples.

I hope this information has been of help. If you would prefer to reply in Spanish again please do, our Spanish speaking Scientific Support specialist returned to the office yesterday and would be happy to help.

I look forward to receiving your reply.

Read More

Answer

Thank you for contacting us and your interest in our products.

Unfortunately, we do not have a direct alternative for the Anti-Proteasome 20S alpha + beta antibody (ab22673). This antibody detects a combination of alpha and beta proteasome subunits. The only other antibodies we have are directed against the alpha subunit (such as mouse monoclonal https://www.abcam.com/Proteasome-20S-alpha-1-2-3-5-6-7-antibody-MCP231-ab22674.htmlor rabbit monoclonal https://www.abcam.com/Proteasome-20S-alpha-2-antibody-EPR5453-ab109525.html) or beta subunit (rabbit polyclonal https://www.abcam.com/Proteasome-20S-beta-6-antibody-C-terminal-ab135537.htmlor mouse monoclonal https://www.abcam.com/Proteasome-20S-beta-6-antibody-ab58081.html).

If you would like to describe in a bit more detail what part of the proteasome you are hoping to detect, I can try to help choose an appropriate alternative. Otherwise, all our antibodies directed towards Proteasome 20S can be found from the following link:

https://www.abcam.com/index.html?pageconfig=searchresults&search=Proteasome 20S&pt=1&sd=2&fViewMore=1

I hope this information has been of some help. If you have any further questions, please do not hesitate to ask.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - other (kidney)
Loading amount
20 µg
Specification
kidney
Gel Running Conditions
Reduced Denaturing (4-12 %)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Jul 11 2012

Answer

Thank you for your reply.

The credit note ID is ***.

I am sorry that this antibody did not perform as stated on the datasheet. I have asked our Finance department to issue a credit note for you. The credit note may be used in one of the following ways:

(1) Redeemed against the original invoice if this hasn't already been paid.
(2) Held on the account for use against a future order.
(3) A full refund can be offered where no other invoices are outstanding.

Please contact your Finance department to confirm how you would like the credit note to be used and ensure it is not redeemed without your knowledge.

To specifically receive a refund please ask your Finance department to contact our Finance department at creditcontrol@abcam.com or by telephone using the information at the “Contact Us” link in the top right corner of our website.

Please refer to the credit note ID in any correspondence with our accounting department. We will send you the completed credit note by email or postal mail with the actual credit note number which will start with the letters CGB.

Let me know if you have any questions or ifthere is anything else that we can do for you, and I'll be happy to help you further.

Read More

Answer

Thank you for your reply and for sending the additional information.

I would be happyto issue a credit note for the customer. Do you have the order number or PO number that this was ordered on? I found one going to Interlab from February 7, order # ***. Is this the correct order?

I look forward to hearing from you and resolving this promptly. Have a great day!

Read More

Question

Dear technical support team:

This customer has purchased ab22673 (Anti-Proteasome 20S alpha + beta antibody) and has conducted the IF assay with rat sample sever times. The results show non-specific staining; therefore, she wants to ask for your help to modify her experiment step, could you please offer any suggestion to help her?

I also attached her data in this letter and her experiment step as follow:

1) Order details:

˙Batch number (Lot number): GR68842-1

˙Abcam product code: ab22673

PO: 1028918

˙Antibody storage conditions (temperature/reconstitution etc) : store at -20°C



2) Please describe the problem (high background, non-specific signal…etc).

Strong non-specific fibrinous staining pattern.



3) On what material are you testing the antibody in IHC/ICC?

˙Species: SD rat

˙What’s cell line or tissue: hippocampal neurons and cortical neurons



4) Sample preparation:

˙Type of sample (Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed

˙paraffin embedded sections, cells in culture, other:____) :

˙Sample preparation: fresh primary neuron culture

˙Positive control:, a beta subunit-specific fluorescence probe (experiments performed with the same set of samples and it showed clear and correct bands in PA gels.)

˙Negative control :



5) Fixation step

˙Yes or No : yes

˙If yes, Fixative agent and concentration: for immunostaining, 4% PFA fixation

˙Fixation time: 12 min

˙Fixation temperature : RT



6) Antigen retrieval method(including time, temperature etc.):



7) Permeabilization method:

˙Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers? Yes.

˙Permeabilizing agent and concentration:



for immunostaining, 0.3% triton-X100 in PBS for 12 min at RT.





8) Blocking agent (eg BSA, serum…):

˙Concentration: 5% BSA

˙Blocking time : 3 hours (trial 1), overnight (trial 2)

˙Blocking temperature: room temp, 4°C



9)

˙Endogenous peroxidases blocked?

˙Endogenous biotins blocked?



10) Primary antibody (If more than one was used, describe in “additional notes”) :

˙Species: rabbit

˙Reacts against: Mouse, Rat, Human, Saccharomyces cerevisiae

˙At what dilution(s) have you tested this antibody: 1:2000, 1:1000

˙Diluent buffer: TBS, 0.1% TBS/T

˙Incubation time: overnight

˙Incubation temperature: 4°C

˙What washing steps were done (which buffer, number of washes): TBS, 0.1% TBS/T; changed buffer at least 4 times during 2 hours



11) Secondary antibody:

˙Species:goat

˙Reacts against which species: rabbit

˙At what dilution(s) have you tested this antibody: 1:5000

˙Diluent buffer: TBS, 0.1% TBS/T

˙Incubation time: 2 hours

˙What washing steps were done (which buffer, number of washes): TBS, 0.1% TBS/T; changed buffer at least times during 2 hours

˙Fluorochrome or enzyme conjugate (eg: FITC, HRP, AP, biotin…etc): HRP

˙Do you know whether the problems you are experiencing come from the secondary?



12) Signal amplification method (eg: ABC, LSAB, HRP polymer, TSE):



13) Detection method (eg: DAB, BCIP/NBT …etc): developed using ECL



14)

˙ How many times have you run this staining? 2 trials

˙Do you obtain the same results every time? yes

˙What steps have you altered to try and optimize the use of this antibody? Lowered the dilution factor of primary antibody and used detergent to reduce background



Could you please help this customer to solve the problem?

Thanks for your kindly help

Best regards

Read More
Answer

Thank you for contacting us and forwarding the customer's protocol details.

I do have a few additional questions, so that I better understand the situation.

The secondary is listed as HRP conjugated, and the detection system is listed as ECL, however the image shows fluorescent deteciton.Is the proteasome stainingin the green or red channel? What other protein has been stained, and what kind of antibody was used?

I've looked through some literature about the expression of this protein, and the subunits can be found in the nucleus or cytoplasm. What kind of staining does the customer expect to see in these hippocampal and cortical neurons?

The customer's protocol looks good, but to reduce background I would recommend blocking the cells with 10% serum from the host of the secondary antibody (10% goat serum in this case) which often improves the results. We do fully guarantee this antibody to work in rat ICC, so if the customer would prefer, I'd be happy to send a replacement or issue a credit or refund.

I look forward to hearing from you. Please let me know if the customer has any further questions, or if there is anything else that we can do for you.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Tissue lysate - whole (Prefrontal cortex)
Loading amount
20 µg
Specification
Prefrontal cortex
Gel Running Conditions
Non-reduced Denaturing (10% acrylamide)
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 27°C

Abcam user community

Verified customer

Submitted Nov 11 2011

Answer

DISCOUNT CODE: *** Expiration date: February 20th, 2012 I am very pleased to hear you would like to accept our offer and test ab22673 in rat. This code will give you 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview for rat 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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1-10 of 14 Abreviews or Q&A

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