Antibodies, Proteins, Kits and Reagents for Life Science

321 638 ₸ Product size

100 µl 321 638 ₸ 10 µl 110 563 ₸

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Anti-ATF6 antibody [EPR22690-84] - ChIP Grade (ab227830)

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [EPR22690-84] to ATF6 - ChIP Grade
Suitable for: WB, IHC-P, ChIP, IP
Knockout validated
Reacts with: Mouse, Human

Product name

Anti-ATF6 antibody [EPR22690-84] - ChIP Grade
See all ATF6 primary antibodies
Description

Rabbit monoclonal [EPR22690-84] to ATF6 - ChIP Grade
Host species

Rabbit

Tested Applications & Species

Application	Species
ChIP	Mouse Human
IHC-P	Human
IP	Human
WB	Human

See all applications and species data

Immunogen

Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
Positive control
- WB: HAP1 and HeLa whole cell lysates. IHC-P: Human kidney tissue. IP: HeLa whole cell lysate. ChIP: Chromatin prepared from RAW 264.7 (treated with tunicamycin) and HeLa (treated with thapsigargin) cells.
General notes
This product is a recombinant monoclonal antibody, which offers several advantages including:
- - High batch-to-batch consistency and reproducibility
- - Improved sensitivity and specificity
- - Long-term security of supply
- - Animal-free production
For more information see here.
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

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: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

EPR22690-84
Isotype

IgG
Research areas

Western blot - Anti-ATF6 antibody [EPR22690-84] (ab227830)

All lanes : Anti-ATF6 antibody [EPR22690-84] - ChIP Grade (ab227830) at 1/1000 dilution

Lane 1 : Wild type HAP1 whole cell lysate 20 µg
Lane 2 : ATF6 knockout HAP1 whole cell lysate 20 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 74 kDa
Observed band size: 90 kDa
why is the actual band size different from the predicted?

ab227830 was shown to specifically react with ATF6 in wild-type HAP1 cells as signal was lost in ATF6 knockout cells. Wild-type and ATF6 knockout samples were subjected to SDS-PAGE. ab227830 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/20,000 dilution for 1 hour at room temperature before imaging.

Exposure time 62 seconds.

Blocking/Diluting buffer and concentration: 5% NFDM/TBST.
Western blot - Anti-ATF6 antibody [EPR22690-84] (ab227830)

All lanes : Anti-ATF6 antibody [EPR22690-84] - ChIP Grade (ab227830) at 1/1000 dilution

Lane 1 : Untreated HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HeLa treated with 1 µ? thapsigargin for 1 hour, whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 74 kDa
Observed band size: 50,90 kDa why is the actual band size different from the predicted?

ATF6 is cleaved upon ER stress and the molecular weight observed is consistent with what has been described in the literature (PMID: 25149687; 11163209).

Exposure time 3 minutes.

Blocking and diluting buffer and concentration: 5% NFDM/TBST.
Immunoprecipitation - Anti-ATF6 antibody [EPR22690-84] (ab227830)

ATF6 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate with ab227830 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab227830 1/1000 dilution (0.5 μg/ml). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg

Lane 2: ab254324 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab227830 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 min.

Lysate were made freshly and used in IP test immediately to minimize protein degradation. Incubation time was 2h.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ATF6 antibody [EPR22690-84] (ab227830)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling ATF6 with ab227830 at 1/1000 dilution (0.566 μg/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on human kidney (PMID: 25725420) is observed. The section was incubated with ab227830 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution 2) for 20mins.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).
ChIP - Anti-ATF6 antibody [EPR22690-84] (ab227830)

Chromatin was prepared from HeLa cells treated with thapsigargin (1 uM, 1 h) according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30 mins and then formaldehyde for 10 min.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab227830 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
Primers and probes are from paper PMID: 17535801.

*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol
ChIP - Anti-ATF6 antibody [EPR22690-84] (ab227830)

Chromatin was prepared from RAW 264.7 cells treated with tunicamycin (5 ug/ml, 4 h) according to the Abcam Dual-X-ChIP protocol*. Cells were fixed with 1.5 mM EGS for 30 mins and then formaldehyde for 10 min.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab227830 (red), or 5 µg of rabbit normal IgG ab172730 (gray) and 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
Primers and probes are from paper PMCID: PMC5179193.

*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol
Anti-ATF6 antibody [EPR22690-84] - ChIP Grade (ab227830)

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