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

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Immunoprecipitation of TAP2 protein from HDLM-2 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is TAP2 (E8G5I) Rabbit mAb. Western blot analysis was performed using TAP2 (E8G5I) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
Simple Western™ analysis of lysates (0.1 mg/mL) from HepG2 cells using Calnexin (C5C9) Rabbit mAb #2679. The virtual lane view (left) shows the target bands (as indicated) at 1:50 and 1:250 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:50 (blue line) and 1:250 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ ​​​​​​​Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Western blot analysis of extracts from various cell lines with IFNGR1 (E444) Antibody (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from SH-SY5Y and KNRK cells using Calreticulin (D3E6) XP® Rabbit mAb.
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.
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)).
Western blot analysis of extracts from HeLa and SW620 cells, untreated (-) or treated with Human Interferon-γ (hIFN-γ) #80385 (100 ng/mL, 48 hr; +), using TAP2 (E8G5I) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower). TAP2 protein expression is induced by IFN-γ treatment, as expected.
Western blot analysis of extracts from PANC1, HepG2 and A204 cells using Calnexin (C5C9) Rabbit mAb.
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).
Western blot analysis of extracts from HeLa and SW620 cells, untreated (-) or treated with Human Interferon-γ (hIFN-γ) #80385 (100 ng/ml, 48 hr; +), using TAP1 (E4T4F) Rabbit mAb (upper) and β-Actin (D6A8) Rabbit mAb #8457 (lower).
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.
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).
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).
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).
Immunohistochemical analysis of paraffin-embedded HeLa (left) and DLD-1 (right) cell pellets using β2-microglobulin (D8P1H) Rabbit mAb.
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).
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human TAP1 (hTAP1-Myc/DDK; +) and Myc/DDK-tagged full-length human TAP2 (hTAP2-Myc/DDK; +), using TAP2 (E8G5I) Rabbit mAb (upper), DYKDDDDK Tag (D6W5B) Rabbit mAb #14793 (middle), or β-Actin (D6A8) Rabbit mAb #8457 (lower).
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).
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.
Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with constructs expressing Myc/DDK-tagged full-length human TAP1 protein (hTAP1-Myc/DDK; +) and Myc/DDK-tagged full-length human TAP2 protein (hTAP2-Myc/DDK; +), using TAP1 (E4T4F) Rabbit mAb (upper), DYKDDDDK Tag Antibody #2368 (middle), and β-Actin (D6A8) Rabbit mAb #8457 (lower).
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).
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.
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.
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).
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).
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).
Immunoprecipitation of TAP1 protein from extracts of HeLa cells treated with Human Interferon-γ (hIFN-γ) #80385 (100 ng/mL, 48 hr). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is TAP1 (E4T4F) Rabbit mAb. Western blot analysis was performed using TAP1 (E4T4F) Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as a secondary antibody.
Immunohistochemical analysis of paraffin-embedded JEG-3 cell pellet (left, positive) or LNCaP cell pellet (right, negative) using HLA-G (E8N9C) XP® Rabbit mAb.
Immunohistochemical analysis of paraffin-embedded human skin using β2-microglobulin (D8P1H) Rabbit mAb.
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).
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).
Immunohistochemical analysis of paraffin-embedded human lung adenocarcinoma using HLA-G (E8N9C) XP® Rabbit mAb.
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.
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To Purchase # 36064
Cat. # Size Qty. Price Inventory
36064T		 1 Kit  (9 x 20 microliters)

Custom Formulation

Product Includes Quantity Applications Reactivity MW(kDa) Isotype
Anti-rabbit IgG, HRP-linked Antibody 7074 100 µl
  • WB
 Rab 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
 All 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
PSMB8/LMP7 (D1K7X) Rabbit mAb 13635 20 µl
  • WB
 H M R 23, 28 Rabbit IgG
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 
TAP2 (E8G5I) Rabbit mAb 25657 20 µl
  • WB
  • IP
 H 72 Rabbit IgG
TAP1 (E4T4F) Rabbit mAb 49671 20 µl
  • WB
  • IP
 H 68 Rabbit IgG

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 (E8G5I) Rabbit mAb recognizes endogenous levels of total TAP2 protein. This antibody does not cross-react with TAP1 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 (E4T4F) Rabbit mAb recognizes endogenous levels of total TAP1 protein. This antibody does not cross-react with TAP2 protein. 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 either with a recombinant protein specific to the carboxy terminus of human TAP1 protein or 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, Val485 of human TAP2 protein, or Pro52 of human calnexin protein. Polyclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Glu444 of human IFNGR1 protein. Polyclonal antibody is 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).

Limited Uses

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For Research Use Only. Not for Use in Diagnostic Procedures.
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U.S. Patent No. 7,429,487, foreign equivalents, and child patents deriving therefrom.
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