R1016

antibody from Acris Antibodies GmbH

Targeting: CIITA C2TA, MHC2TA, NLRA

Western blot (Supportive data in Antibodypedia)

ELISA (Recommended by provider)

Immunoprecipitation (Recommended by provider)

Antibody Data

Product number

R1016

Provider

Acris Antibodies GmbH

Proper citation

Acris Antibodies GmbH Cat#R1016, RRID:AB_977979

Product name

anti CIITA / C2TA (1-333)

Antibody type

Polyclonal

Antigen

Peptide corresponding to a region near the N-terminal end of the human CIITA gene conjugated to Keyhole Limpet Hemocyanin (KLH).

Reactivity

Human

Host

Rabbit

Vial size

0.1 ml

Concentration

90.0 mg/ml (by Refractometry)

Western blot

Supportive validation

Submitted by

Acris Antibodies GmbH (provider)

Main image

Experimental details

Figure 1. Immunoblot of anti-CIITA antibodies shows specificity.Anti-CIITA (1-333) antibody, generated by immunization with bacterially producedFLAG-CIITA aa 1-333, was tested by immunoblot against lysates of Cos-7 cells after transient transfection, separately, with pcDNA3-FLAG-CIITA-336-884 and pcDNA3-HA-CIITA. For transfection, Fugene 6 (Roche) was used according to themanufacturer's instructions for a 6-well plate format. Cells were lysed 24 h posttransfectionin 200 µL of 1x SDS-sample buffer, heated at 96°C for 5', and vortexed for 30 sec. Samples (10 µL each) were separated on a 12% SDS-PAGE gel andtransferred to PVDF (Millipore) followed by blocking for 45' using TTBS supplemented with 5% non-fat dry milk. All incubations were performed at room temperature.In panel A, both samples on PVDF were incubated with 10 µg/mL mouse anti-FLAG antibody (Sigma) for 45'. After 5X washes with TTBS, reaction with ALP rabbit antimouse IgG at 200 ng/mL proceeded for 45' following again by washing as before.The blot was developed using BCIP/NBT. This blot demonstrates that FLAGCIITA-336-842 was successfully over-expressed in the Cos-7 cells.In panel B, both samples on PVDF were incubated with a 1:500 dilution of Rockland's anti-CIITA (1-333) for 45'. After 5X washes with TTBS,reaction with HRP goat anti-rabbit IgG at 10 ng/mL proceeded for 45' following again by washing as before. The membrane was covered withPico West Substrate solution (Pierce) for 5' and was then placed between the two layers of a standard sheet protector. Kodak O-MAT filmwas exposed to the blot for 30 sec and was immediately developed. The lane containing the lysate of pcDNA3-HA-CIITA transfected cellscontains a single band at ~130 kD molecular weight, whereas the lane containing lysate from pcDNA3-FLAG-CIITA-336-842 transfected cellsshows no reactivity.

Supportive validation

Submitted by

Acris Antibodies GmbH (provider)

Main image

Experimental details

Figure 2. Immunoblot of anti-CIITA antibodies shows sensitivity.Anti-CIITA (1-333) antibody, generated by immunization with bacteriallyproduced FLAG-CIITA aa 1-333, was tested by immunoblot against lysatesfrom Spodoptera frugiperda 21 (SF21) cells (2 x 105 cells in 200 µL of 1x SDSsamplebuffer) and an SF21 lysate spiked with recombinant FLAG-CIITA to 0.2ng/µL. Cells from both preparations were lysed by heating at 96°C for 5' andvortexing for 30 sec. Samples (5 µL each) were separated on a 12% SDSPAGEgel either stained with Coomassie Brilliant Blue or transferred to PVDF(Millipore) followed by blocking for 45' using TTBS supplemented with 5% nonfatdry milk. All incubations were performed at room temperature.In panel A, both samples of SF21 lysate and the SF21 lysate supplementedwith CIITA are stained with Coomassie Brilliant Blue. The similar intensity ofstaining in each lane indicates that ~ equal amounts of total protein arepresent in both preparations. No band at the molecular weight of CIITA (130KD) is distinguishable in the SF21 lysate supplemented with recombinantCIITA.In panel B, both samples on PVDF were incubated with a 1:500 dilution of Rockland's anti-CIITA (1-333) for 45'. After 5X washes withTTBS, reaction with HRP goat anti-rabbit IgG at 10 ng/mL proceeded for 45' following again by washing as before. The membrane wascovered with Pico West Substrate solution (Pierce) for 5' and was then placed between the two layers of a standard sheet protector.Kodak O-MAT film was exposed to the blot for 30 sec and was immediately developed. The lane containing the lysate of SF21 spikedwith 1 ng of recombinant FLAG-CIITA (1-333) is clearly detectable as a single band at ~130 kD molecular weight, whereas no staining isevident in the control lysate of SF21 cells. This antibody may detect levels of CIITA below 1 ng when other detection techniques areused.