Overview

  • Product name
    Anti-HLA-DR antibody [L243]
    See all HLA-DR primary antibodies
  • Description
    Mouse monoclonal [L243] to HLA-DR
  • Host species
    Mouse
  • Specificity
    ab136320 recognizes specifically HLA-DR molecules both peptide-loaded and empty.
  • Tested applications
    Suitable for: WB, IP, IHC-P, IHC-Fr, Functional Studies, ICC, Flow Cyt, Blockingmore details
  • Species reactivity
    Reacts with: Dog, Human, Non human primates
  • Immunogen

    Tissue, cells or virus corresponding to HLA-DR. Human B lymphocytes.

  • Positive control
    • Flow Cytometry: Human peripheral blood cells.

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 long term. Avoid freeze / thaw cycle.
  • Storage buffer
    pH: 7.40
    Preservative: 0.1% Sodium azide
    Constituent: PBS
  • Concentration information loading...
  • Purity
    Protein A purified
  • Purification notes
    Purified from cell culture supernatant by protein A affinity chromatography. Purity: > 95% (by SDS-PAGE).
  • Clonality
    Monoclonal
  • Clone number
    L243
  • Isotype
    IgG2a
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab136320 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 at an assay dependent concentration. Predicted molecular weight: 29 kDa.
IP Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.
IHC-Fr Use at an assay dependent concentration.
Functional Studies Use at an assay dependent concentration.
ICC Use at an assay dependent concentration.
Flow Cyt Use 0.1µg for 106 cells.

ab170191 - Mouse monoclonal IgG2a, is suitable for use as an isotype control with this antibody.

 

Blocking Use at an assay dependent concentration.

Target

  • Function
    Binds peptides derived from antigens that access the endocytic route of antigen presenting cells (APC) and presents them on the cell surface for recognition by the CD4 T-cells. The peptide binding cleft accommodates peptides of 10-30 residues. The peptides presented by MHC class II molecules are generated mostly by degradation of proteins that access the endocytic route, where they are processed by lysosomal proteases and other hydrolases. Exogenous antigens that have been endocytosed by the APC are thus readily available for presentation via MHC II molecules, and for this reason this antigen presentation pathway is usually referred to as exogenous. As membrane proteins on their way to degradation in lysosomes as part of their normal turn-over are also contained in the endosomal/lysosomal compartments, exogenous antigens must compete with those derived from endogenous components. Autophagy is also a source of endogenous peptides, autophagosomes constitutively fuse with MHC class II loading compartments. In addition to APCs, other cells of the gastrointestinal tract, such as epithelial cells, express MHC class II molecules and CD74 and act as APCs, which is an unusual trait of the GI tract. To produce a MHC class II molecule that presents an antigen, three MHC class II molecules (heterodimers of an alpha and a beta chain) associate with a CD74 trimer in the ER to form an heterononamer. Soon after the entry of this complex into the endosomal/lysosomal system where antigen processing occurs, CD74 undergoes a sequential degradation by various proteases, including CTSS and CTSL, leaving a small fragment termed CLIP (class-II-associated invariant chain peptide). The removal of CLIP is facilitated by HLA-DM via direct binding to the alpha-beta-CLIP complex so that CLIP is released. HLA-DM stabilizes MHC class II molecules until primary high affinity antigenic peptides are bound. The MHC II molecule bound to a peptide is then transported to the cell membrane surface. In B-cells, the interaction between HLA-DM and MHC class II molecules is regulated by HLA-DO. Primary dendritic cells (DCs) also to express HLA-DO. Lysosomal miroenvironment has been implicated in the regulation of antigen loading into MHC II molecules, increased acidification produces increased proteolysis and efficient peptide loading.
  • Sequence similarities
    Belongs to the MHC class II family.
    Contains 1 Ig-like C1-type (immunoglobulin-like) domain.
  • Post-translational
    modifications
    Ubiquitinated by MARCH1 or MARCH8 at Lys-244 leading to down-regulation of MHC class II. When associated with ubiquitination of the beta subunit of HLA-DR: HLA-DRB4 'Lys-254', the down-regulation of MHC class II may be highly effective.
  • Cellular localization
    Cell membrane. Endoplasmic reticulum membrane. Golgi apparatus > trans-Golgi network membrane. Endosome membrane. Lysosome membrane. Late endosome membrane. The MHC class II complex transits through a number of intracellular compartments in the endocytic pathway until it reaches the cell membrane for antigen presentation.
  • Information by UniProt
  • Database links
  • Alternative names
    • DR alpha chain antibody
    • DR alpha chain precursor antibody
    • DRA_HUMAN antibody
    • DRB1 antibody
    • DRB4 antibody
    • Histocompatibility antigen HLA DR alpha antibody
    • HLA class II histocompatibility antigen antibody
    • HLA class II histocompatibility antigen DR alpha chain antibody
    • HLA DR1B antibody
    • HLA DR3B antibody
    • HLA DRA antibody
    • HLA DRA1 antibody
    • HLA DRB1 antibody
    • HLA DRB3 antibody
    • HLA DRB4 antibody
    • HLA DRB5 antibody
    • HLA-DRA antibody
    • HLADR4B antibody
    • HLADRA1 antibody
    • HLADRB antibody
    • Major histocompatibility complex class II DR alpha antibody
    • Major histocompatibility complex class II DR beta 1 antibody
    • Major histocompatibility complex class II DR beta 3 antibody
    • Major histocompatibility complex class II DR beta 4 antibody
    • Major histocompatibility complex class II DR beta 5 antibody
    • MGC117330 antibody
    • MHC cell surface glycoprotein antibody
    • MHC class II antigen DRA antibody
    • MHC II antibody
    • MLRW antibody
    see all

Images

  • Flow Cytometry analysis of human peripheral blood cells labeling HLA DR with ab136320, followed by a Goat anti-mouse-APC secondary.

  • Human peripheral blood lymphocytes stained with ab136320 (red line). Human whole blood was processed using a modified protocol based on Chow et al, 2005 (PMID: 16080188).

    In brief, human whole blood was fixed in 4% formaldehyde (methanol-free) for 10 minutes at 22°C. Red blood cells were then lyzed by the addition of Triton X-100 (final concentration - 0.1%) for 15 minutes at 37°C. For experimentation, cells were treated with 50% methanol (-20°C) for 15 minutes at 4°C. Cells were then incubated with the antibody (ab136320, 0.1μg/1x106 cells) for 30 minutes at 4°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 minutes at 4°C. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 0.1μg/1x106cells) used under the same conditions. Unlabeled sample (blue line) was also used as a control. Acquisition of >30,000 total events were collected using a solid-state 25mW red diode laser (635nm) and 675/30 bandpass filter. Gating strategy - peripheral blood lymphocytes.

References

ab136320 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Application
Western blot
Sample
Human Cell lysate - whole cell (RERF-LC-Ad1 cells)
Gel Running Conditions
Reduced Denaturing
Loading amount
25 µg
Specification
RERF-LC-Ad1 cells
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 4% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Nov 06 2017

Answer

Thank you very much for your reply.

The binding affinity and mechanism varies between blocking antibodies, butthey do seem to generally act as antagonists. These kinds of characteristics need to be determined for each individual antibody and antigen. For the HLA antibody Ab23632, we only have one publication demonstrating its use in a blocking experiment and there doesn't seem to be any data regarding the binding affinity of the antibody-

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1069546/

The specific time the antibody will bind to a single antigen depends on factors of the experiment such as temperature, concentration of the antibody in the media, cell type, etc. I wouldn't expect the blocking to be effective for many days, but probably the effect will be noticeable on the hour-scale.

I am sorry that we don't havemore information regarding this, but if you have further questions or need anything else, please let me know and I'll be happy to help.

Read More

Answer

Thank you again for contacting us and for your patience while I've been in touch with the lab about your enquiry.

Ab20897 against HLA-DP has not been tested in a blocking experiment at this time, though it is possible that it will work in such an assay. Unfortunately none of our other HLA-DP antibodies seem tohave been tested for blocking ability atthis time. If you're interested in trying Ab20897 in a blocking experiment, we do have a testing discount programand I'd be happy to send more information about this if you're interested.

The clone SPV-L3 against HLA-DQ, which we carry as Ab23632, has been used in a blocking experiment in the following publication-

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1069546/

We would recommend removing the sodium azide from Ab23632 before incubating it with live cells, as azide will cause cell death. An antibody purification kit such as Ab102784 can be used to remove the azide-

https://www.abcam.com/Antibody-Purification-Kit-Protein-A-ab102784.html

I hope that this information is useful, but please let me know if you have any further questions and I'll be happy to help.

Read More

Answer

Thank you very much for contacting us with your enquiry.

Theterm "functional studies" can be used to describe several different kinds of assays, and sometimes blocking assays are included in this categorization so there isn't a specific difference between blocking and functional studies antibodies.

For example, ab136320 is listed as being tested in functional studies and a blocking assay, however after checking through our internal notes I see that the functional study was actually a blocking assay. Ab23755 was also tested in a functional study, however this was a tissue typing and crossmatching experiment,not a blocking assay. The details of the functional study are often on the datasheet in the "Application Notes" section, however this is not always the case. I apologize that these designationsare not more straight-forward and clear.

Are you using human samples? Ab136320 will be a good antibody for blocking HLA DR, but we don't currently have any antibodies listed as being tested for blocking of HLA DP and HLA DQ. There are two antibodies to these targets that might work in such an assay, ab20897 (HLA DP) and ab23632 (HLA DQ), but I'm checking with the lab to make sure they've been tested for blocking ability. I'll get back to you as soon as I have confirmed this.

I hope that this information will be useful, but please let me know if you have any further questions and I'll be happy to help.

Read More

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