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36064
MHC Class I Antigen Processing and Presentation Antibody Sampler Kit
Primary Antibodies
Antibody Sampler Kit

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit #36064

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MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 1

After the primary antibody is bound to the target protein, a complex with HRP-linked secondary antibody is formed. The LumiGLO® is added and emits light during enzyme catalyzed decomposition.

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 2

Flow cytometric analysis of Jurkat cells using Calreticulin (D3E6) XP® Rabbit mAb (solid line) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (dashed line). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 3

Western blot analysis of extracts from HeLa, NIH/3T3, and H-4-II-E cells, untreated (-) or treated with MG-132 #2194 (10μM, 90min; +), using Ubiquitin (E4I2J) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 4

Immunohistochemical analysis of paraffin-embedded JEG-3 cell pellet (left, positive) or LNCaP cell pellet (right, negative) using HLA-G (E8N9C) XP® Rabbit mAb.

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 5

Confocal immunofluorescent analysis of HeLa cells using Calnexin (C5C9) Rabbit mAb (green). Actin filaments have been labeled using DY-554 phalloidin (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 6

Immunoprecipitation of TAP2 from HeLa cell extracts treated with Human Interferon-γ (hIFN-γ) #8901 (10 ng/ml, 16 hr), using Normal Rabbit IgG #2729 (lane 2) or TAP2 Antibody (lane 3). Lane 1 is 10% input. Western blot analysis was performed using TAP2 Antibody.

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 7

Western blot analysis of extracts from various cell lines using PSMB8/LMP7 (D1K7X) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). (The T2 cell line contains a homozygous deletion of PSMB8/LMP7 (13)).

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 8

Western blot analysis of extracts from HeLa and HT-29 cells, untreated (-) or treated with Human Interferon-γ (hIFN-γ) #8901 (10 ng/ml, 16 hr; +), using TAP1 Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 9

Flow cytometric analysis of DLD-1 cells (blue) and HeLa cells (green) using β2-microglobulin (D8P1H) Rabbit mAb. Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 647 Conjugate) #4414 was used as a secondary antibody.

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 10

Immunoprecipitation of IFNGR1 from NCI-H226 cell extracts. Lane 1 is 10% input, lane 2 is Normal Rabbit IgG #2729, and lane 3 is IFNGR1 (E444) Antibody. Western blot analysis was performed using IFNGR1 (E444) Antibody. Mouse Anti-rabbit IgG (Conformation Specific) (L27A9) mAb #3678 was used for detection.

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 11

Confocal immunofluorescent analysis of NIH/3T3 cells using Calreticulin (D3E6) XP® Rabbit mAb (green) and β-Actin (13E5) Rabbit mAb (Alexa Fluor® 555 Conjugate) #8046 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 12

Western blot analysis of various recombinant linkage-specific polyubiquitin chains, recombinant free monoubiquitin (MonoUb), and recombinant linear polyubiquitin (Ub linear) (300 ng each), using Ubiquitin (E4I2J) Rabbit mAb.

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 13

Immunohistochemical analysis of paraffin-embedded human placenta using HLA-G (E8N9C) XP® Rabbit mAb (left) compared to concentration matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right).

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 14

Immunohistochemical analysis of paraffin-embedded human breast carcinoma using Calnexin (C5C9) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 15

Western blot analysis of extracts from HeLa and HT-29 cells, untreated (-) or treated with Human Interferon-γ (hIFN-γ) #8901 (10 ng/ml, 16 hr; +), using TAP2 Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 16

Western blot analysis of extracts from HeLa and SW620 cells, untreated (-) or treated with Human Interferon-γ (hIFN-γ) #8901 (100 ng/ml, 72 hr; +), using PSMB8/LMP7 (D1K7X) Rabbit mAb (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower).

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 17

Confocal immunofluorescent analysis of PANC-1 (positive, left) and DLD-1 (negative, right) cells, using β2-microglobulin (D8P1H) Rabbit mAb (green). Blue pseudocolor= DRAQ5® #4084 (fluorescent DNA dye).

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 18

Western blot analysis of extracts from various cell lines with IFNGR1 (E444) Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 19

Western blot analysis of extracts from SH-SY5Y and KNRK cells using Calreticulin (D3E6) XP® Rabbit mAb.

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 20

Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma using HLA-G (E8N9C) XP® Rabbit mAb.

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 21

Western blot analysis of extracts from PANC1, HepG2 and A204 cells using Calnexin (C5C9) Rabbit mAb.

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 22

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human PSMB5 (hPSMB5-Myc/DDK; +) or Myc/DDK-tagged full-length human PSMB8 (hPSMB8-Myc/DDK; +), using PSMB8/LMP7 (D1K7X) Rabbit mAb (upper) and DYKDDDDK Tag Antibody #2368 (lower).

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 23

Immunohistochemical analysis of paraffin-embedded HeLa (left) and DLD-1 (right) cell pellets using β2-microglobulin (D8P1H) Rabbit mAb.

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 24

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human IFNGR1 protein (hIFNGR1-Myc/DDK; +), using IFNGR1 (E444) Antibody (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 25

Western blot analysis of extracts from JEG-3 and LNCaP cells using HLA-G (E8N9C) XP® Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 26

Immunohistochemical analysis of paraffin-embedded colon carcinoma using β2-microglobulin (D8P1H) Rabbit mAb in the presence of control peptide (left) or antigen-specific peptide (right).

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 27

Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with construct expressing full-length human MycDDK-tagged HLA-G (hHLA-G-MycDDK; +), using HLA-G (E8N9C) XP® Rabbit mAb (upper), Myc-Tag (71D10) Rabbit mAb #2278 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 28

Immunohistochemical analysis of paraffin-embedded human skin using β2-microglobulin (D8P1H) Rabbit mAb.

MHC Class I Antigen Processing and Presentation Antibody Sampler Kit: Image 29

Western blot analysis of extracts from various cell lines using β2-microglobulin (D8P1H) Rabbit mAb (upper) and β-Actin (DA8) Rabbit mAb #8457 (lower). DLD-1 and Daudi cell lines are negative for β2-microglobulin due to genomic deletions at the β2-microglobulin locus.

To Purchase # 36064T
Product # Size Price
36064T
1 Kit  (9 x 20 µl) $ 609

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
Goat 
Calreticulin (D3E6) XP® Rabbit mAb 12238 20 µl
  • WB
  • IF
  • F
H M R 55 Rabbit IgG
Ubiquitin (E4I2J) Rabbit mAb 43124 20 µl
  • WB
H M R 9-300 Rabbit IgG
HLA-G (E8N9C) XP® Rabbit mAb 79769 20 µl
  • WB
  • IHC
H 30-40 Rabbit IgG
Calnexin (C5C9) Rabbit mAb 2679 20 µl
  • WB
  • IHC
  • IF
H Mk 90 Rabbit IgG
TAP2 Antibody 12259 20 µl
  • WB
  • IP
H 72 Rabbit 
PSMB8/LMP7 (D1K7X) Rabbit mAb 13635 20 µl
  • WB
H M R 23, 28 Rabbit IgG
TAP1 Antibody 12341 20 µl
  • WB
H M 68 Rabbit 
β2-microglobulin (D8P1H) Rabbit mAb 12851 20 µl
  • WB
  • IHC
  • IF
  • F
H Mk 12 Rabbit IgG
IFNGR1 (E444) Antibody 10405 20 µl
  • WB
  • IP
H 45-90 Rabbit 

Product Description

The MHC Class I Antigen Processing and Presentation Antibody Sampler Kit provides an economical means to examine key proteins associated with the processing and presentation of MHC class I-restricted antigens. The provided antibodies allow monitoring of total protein levels. The kit includes enough antibodies to perform two western blot experiments with each primary antibody.

Specificity / Sensitivity

Each antibody in the MHC Class I Antigen Processing and Presentation Antibody Sampler Kit detects endogenous levels of its target protein. Calreticulin (D3E6) XP® Rabbit mAb recognizes endogenous levels of total calreticulin protein. Ubiquitin (E4I2J) Rabbit mAb recognizes endogenous levels of free ubiquitin and polyubiquitinated proteins. This antibody is able to detect free ubiquitin, linear polyubiquitin (M1-linked), and homotypic polyubiquitin chains consisting of K6, K11, K27, K29, K33, K48 and K63 linkages. HLA-G (E8N9C) XP® Rabbit mAb recognizes endogenous levels of total HLA-G protein. Calnexin (C5C9) Rabbit mAb detects endogenous levels of total calnexin protein. TAP2 Antibody recognizes endogenous levels of total TAP2 protein. PSMB8/LMP7 (D1K7X) Rabbit mAb recognizes endogenous levels of total PSMB8/LMP7 protein. This antibody recognizes both 28 kDa precursor and 23 kDa mature forms of PSMB8/LMP7 and does not cross-react with PSMB5 protein. This antibody recognizes proteins of unknown origin in the 80-100 kDa range. TAP1 Antibody recognizes endogenous levels of total TAP1 protein. This antibody cross-reacts with a 100 kDa protein of unknown origin. β2-microglobulin (D8P1H) Rabbit mAb recognizes endogenous levels of total β2-microglobulin protein. IFNGR1 (E444) Antibody recognizes endogenous levels of total IFNGR1 protein.

Source / Purification

Monoclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the amino terminus of human calreticulin protein, the carboxy terminus of human PSMB8/LMP7 protein, Gly35 of human ubiquitin protein, Leu102 of human HLA-G protein, Val57 of human β2-microglobulin protein, and Pro52 of human calnexin protein. Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val612 of mouse TAP1 protein, Phe588 of human TAP2 protein, and Glu444 of human IFNGR1 protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography.

Background

The predominant function of class I MHC/β2-microglobulin dimers, which are expressed on the surface of most nucleated cell types, is to modulate the adaptive immune response by presenting proteolytic peptide fragments from cytosolic proteins to cytotoxic CD8+ T cells. In order for self and nonself peptides to be presented by MHC class I molecules, the peptide fragments must first be derived from polyubiquitinated proteins that undergo degradation via the ubiquitin-proteasome system. In the context of inflammatory processes, the enzymatic core of the proteasome can be shaped by IFNγ signaling to contain subunits, such as PSMB8/LMP7, which enhance the presentation of antigenic peptides by antigen presenting cells (1). The resulting cytosolic peptide fragments generated through ubiquitin-dependent proteasomal degradation are then transported into the ER lumen via the peptide transporters, TAP1 and TAP2, where the activity of multiple chaperone proteins, such as calnexin and calreticulin, facilitate loading onto class I MHC/β2-microglobulin dimers for transport to the Golgi and eventually, the cell surface (2-6). Defects in the expression of multiple components of the class I antigen presenting machinery have been observed in both solid and liquid tumors, which serves as a mechanism of tumor-immune evasion (7).

  1. Ferrington, D.A. and Gregerson, D.S. (2012) Prog Mol Biol Transl Sci 109, 75-112.
  2. Antoniou, A.N. et al. (2003) Curr Opin Immunol 15, 75-81.
  3. Jensen, P.E. (2007) Nat Immunol 8, 1041-8.
  4. Kloetzel, P.M. (2001) Nat Rev Mol Cell Biol 2, 179-87.
  5. Sant, A. and Yewdell, J. (2003) Curr Opin Immunol 15, 66-8.
  6. Yewdell, J.W. (2005) Immunol Rev 207, 8-18.
  7. Seliger, B. (2008) Cancer Immunol Immunother 57, 1719-26.

Pathways & Proteins

Explore pathways + proteins related to this product.

Limited Uses

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
XP is a registered trademark of Cell Signaling Technology, Inc.
U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.