Name: Anti-Bad antibody [Y208]
Brand: RabMAb
SKU: ab32445
Rating: 4 (13 reviews)

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Western blot - Anti-Bad antibody [Y208] (ab32445)

Key features and details

Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
Rabbit monoclonal [Y208] to Bad
Suitable for: WB, IHC-P, ICC/IF, Flow Cyt
Knockout validated
Reacts with: Mouse, Rat, Human

Product name

Anti-Bad antibody [Y208]
See all Bad primary antibodies
Description

Rabbit monoclonal [Y208] to Bad
Host species

Rabbit
Specificity

The antibody does not cross-react with other Bcl-2 members.
Tested applications

Suitable for: WB, IHC-P, ICC/IF, Flow Cytmore details
Species reactivity

Reacts with: Mouse, Rat, Human
Predicted to work with: Dog
Immunogen

Synthetic peptide within Human Bad (N terminal). The exact sequence is proprietary.
(Peptide available as ab206866)
Positive control
- WB: HAP1, MCF7 and HeLa cell lysate. IHC-P: Human lymphoma. ICC/IF: RAW 264.7. Flow Cyt: MCF7.
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.
Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.

Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.

We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.

In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.

We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.

Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.

Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.

Form

Liquid
Storage instructions

Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
Storage buffer

pH: 7.20
Preservative: 0.01% Sodium azide
Constituents: 49% PBS, 50% Glycerol (glycerin, glycerine), 0.05% BSA
Concentration information loading...
Purity

Protein A purified
Clonality

Monoclonal
Clone number

Y208
Isotype

IgG
Research areas

Alternative Versions
- Biotin Anti-Bad antibody [Y208] (ab199754)
- Anti-Bad antibody [Y208] - BSA and Azide free (ab220116)
Compatible Secondaries
Isotype control
- Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
KO cell lysates
- Human BAD knockout HeLa cell lysate (ab256847)
Recombinant Protein
- Recombinant Human Bad protein (ab89848)

Our Abpromise guarantee covers the use of ab32445 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		1/2000 - 1/5000. Detects a band of approximately 23 kDa (predicted molecular weight: 18 kDa).
IHC-P		Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF		1/250 - 1/500.
Flow Cyt		1/20 - 1/50. ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

Function

Promotes cell death. Successfully competes for the binding to Bcl-X(L), Bcl-2 and Bcl-W, thereby affecting the level of heterodimerization of these proteins with BAX. Can reverse the death repressor activity of Bcl-X(L), but not that of Bcl-2 (By similarity). Appears to act as a link between growth factor receptor signaling and the apoptotic pathways.
Tissue specificity

Expressed in a wide variety of tissues.
Sequence similarities

Belongs to the Bcl-2 family.
Domain

Intact BH3 motif is required by BIK, BID, BAK, BAD and BAX for their pro-apoptotic activity and for their interaction with anti-apoptotic members of the Bcl-2 family.
Post-translational
modifications

Phosphorylated on one or more of Ser-75, Ser-99, Ser-118 and Ser-134 in response to survival stimuli, which blocks its pro-apoptotic activity. Phosphorylation on Ser-99 or Ser-75 promotes heterodimerization with 14-3-3 proteins. This interaction then facilitates the phosphorylation at Ser-118, a site within the BH3 motif, leading to the release of Bcl-X(L) and the promotion of cell survival. Ser-99 is the major site of AKT/PKB phosphorylation, Ser-118 the major site of protein kinase A (CAPK) phosphorylation. Ser-75 is phosphorylated by AKT/PKB, protein kinase A and PIM2.
Cellular localization

Mitochondrion outer membrane. Cytoplasm. Upon phosphorylation, locates to the cytoplasm.
Target information above from: UniProt accession Q92934 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
Database links
- Entrez Gene: 483763 Dog
- Entrez Gene: 572 Human
- Entrez Gene: 12015 Mouse
- Entrez Gene: 64639 Rat
- Omim: 603167 Human
- SwissProt: Q92934 Human
- SwissProt: Q61337 Mouse
- SwissProt: O35147 Rat
- Unigene: 370254 Human
- Unigene: 4387 Mouse
- Unigene: 36696 Rat
see all
Alternative names
- AI325008 antibody
- BAD antibody
- BAD_HUMAN antibody
- BBC 2 antibody
- BBC2 antibody
- BBC6 antibody
- Bcl 2 Antagonist of Cell Death antibody
- Bcl 2 Binding Component 6 antibody
- BCL X / BCL 2 Binding Protein antibody
- BCL X Binding Protein antibody
- Bcl XL/Bcl 2 Associated Death Promoter antibody
- Bcl-2-binding component 6 antibody
- Bcl-2-like protein 8 antibody
- Bcl-XL/Bcl-2-associated death promoter antibody
- Bcl2 antagonist of cell death antibody
- BCL2 antagonist of cell death protein antibody
- BCL2 associated agonist of cell death antibody
- Bcl2 Associated Death Promoter antibody
- BCL2 binding component 6 antibody
- BCL2 binding protein antibody
- Bcl2 Like 8 Protein antibody
- Bcl2-L-8 antibody
- BCL2L8 antibody
- Proapoptotic BH3 Only Protein antibody
see all

Western blot - Anti-Bad antibody [Y208] (ab32445)

All lanes : Anti-Bad antibody [Y208] (ab32445) at 1/2000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : BAD knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 18 kDa
Observed band size: 23 kDa
why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab32445 observed at 23 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

ab32445 was shown to react with Bad in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab264843 (knockout cell lysate ab256847) was used. Wild-type HeLa and BAD knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32445 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 2000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Bad antibody [Y208] (ab32445)

Immunohistochemical staining of paraffin-embedded human lymphoma using ab32445 at 1/100 dilution.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
Western blot - Anti-Bad antibody [Y208] (ab32445)

All lanes : Anti-Bad antibody [Y208] (ab32445) at 1/2000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : BAD knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : MCF7 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 18 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab32445 observed at 23 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab32445 was shown to specifically recognise BAD in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when BAD knockout cells were examined. Wild-type and BAD knockout samples were subjected to SDS-PAGE. Ab32445 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/2000 dilution and 1/20,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.
Immunocytochemistry/ Immunofluorescence - Anti-Bad antibody [Y208] (ab32445)

Immunocytochemical analysis of 2% paraformaldehyde-fixed,0.1% Triton-X100 permeabilized RAW 264.7 labeling Bad with ab32445 at 1/250dilution,followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 568) secondary antibody at 1/1000 dilution.
Western blot - Anti-Bad antibody [Y208] (ab32445)

Anti-Bad antibody [Y208] (ab32445) at 1/5000 dilution + Hela cell lysate

Predicted band size: 18 kDa
Observed band size: 23 kDa why is the actual band size different from the predicted?
Flow Cytometry - Anti-Bad antibody [Y208] (ab32445)

Overlay histogram showing MCF-7 cells stained with ab32445 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32445, 1/20 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit monoclonal IgG (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in MCF-7 cells fixed with methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.
Anti-Bad antibody [Y208] (ab32445)

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

Datasheet

Publishing research using ab32445? Please let us know so that we can cite the reference in this datasheet.

ab32445 has been referenced in 73 publications.

Li Q et al. Silencing of synaptotagmin 13 inhibits tumor growth through suppressing proliferation and promoting apoptosis of colorectal cancer cells. Int J Mol Med 45:234-244 (2020). PubMed: 31939613
Zheng J et al. MSCs ameliorate hepatocellular apoptosis mediated by PINK1-dependent mitophagy in liver ischemia/reperfusion injury through AMPKa activation. Cell Death Dis 11:256 (2020). PubMed: 32312955
Hu J et al. ERO1a inhibits cell apoptosis and regulates steroidogenesis in mouse granulosa cells. Mol Cell Endocrinol 511:110842 (2020). PubMed: 32376276
Liu K et al. Protective effects of calbindin-D28K on the UVB radiation-induced apoptosis of human lens epithelial cells. Int J Mol Med 45:1793-1802 (2020). PubMed: 32236567
Feng C et al. Neuroprotective Effect of Danhong Injection on Cerebral Ischemia-Reperfusion Injury in Rats by Activation of the PI3K-Akt Pathway. Front Pharmacol 11:298 (2020). PubMed: 32218735

View all Publications for this product

Customer reviews and Q&As

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1-10 of 17 Abreviews or Q&A

Application

Western blot

Sample

Human Cell lysate - whole cell (SKOV-3)

Gel Running Conditions

Reduced Denaturing (10)

Loading amount

20 µg

Specification

SKOV-3

Blocking step

BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C

Abcam user community

Verified customer

Submitted Aug 19 2019

Application

Western blot

Sample

Human Tissue lysate - whole (Heart)

Gel Running Conditions

Reduced Denaturing

Loading amount

25 µg

Specification

Heart

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Dec 24 2018

Application

Western blot

Sample

Rat Tissue lysate - whole (Heart)

Gel Running Conditions

Reduced Denaturing

Loading amount

25 µg

Specification

Heart

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Dec 24 2018

Application

Immunocytochemistry

Sample

Rat Cultured Cells (Cardiomyocytes)

Permeabilization

Yes - Triton x-100, 0.03%

Specification

Cardiomyocytes

Blocking step

BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C

Fixative

Paraformaldehyde

Abcam user community

Verified customer

Submitted Feb 28 2018

Question

Please send the IgG concentrations of the following lots.
ab33906 lot YH090207C -
ab33862 lot YH082410D -
ab32429 lot YG080503 -
ab40660 lot YI070802CS -
ab32445 lot YI072804DS -

Abcam community

Verified customer

Asked on Feb 05 2013

Answer

ab33906 lot YH090207C - 0.3260 mg/ml

ab33862 lot YH082410D - 0.6380 mg/ml

ab32429 lot YG080503 - 0.0790 mg/ml

ab40660 lot YI070802CS - 0.1030 mg/ml

ab32445 lot YI072804DS - 0.0250 mg/ml

Abcam Scientific Support

Answered on Feb 05 2013

Application

Immunocytochemistry

Sample

Mouse Cultured Cells (RAW 264.7)

Specification

RAW 264.7

Fixative

Paraformaldehyde

Permeabilization

Yes - 0.1% Triton-X100 in 2% BSA for 15min

Blocking step

BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 22°C

Dr. Mahesh Shivananjappa

Verified customer

Submitted May 19 2012

Question

Dear Technical service,
Which antibodies could you recommended as a 2nd step to the following products:
ab32445 and ab33862 for flow cytometry?
Awaiting your answer.
Best regards.

Abcam community

Verified customer

Asked on Mar 28 2012

Answer

Thank you for contacting us.
There is a wide range of secondaries suitable to detect either ab32445 or ab33862 in Flow Cytometry. I have conducted a search through our Secondaries search tool in the Abcam Website and found more than 100 matches, depending on the conjugation dye.
I would recommend you to have a look at the results and depending on the wavelength you are interested in measuring, choose the option that better matches your requirements.
https://www.abcam.com/index.html?pageconfig=searchresults&search=IgG&pt=7&sk=app&sv=99&sn=Flow Cyt&l=3&fViewMore=1
If you have any doubt about any of the products identified in the search, you need a proforma, or require further assistance, please do not hesitate to contact me back.

Abcam Scientific Support

Answered on Mar 28 2012

Question

Ordered ab90435 by mistake and would like to return it.

Abcam community

Verified customer

Asked on Feb 09 2012

Answer

Thank you for contacting Abcam.

Included in this email are the instructions for returning your item to Abcam. When you return the goods, please ensure that you follow the procedures as below:

- Return in the original Abcam box with the cooler box and ice pack (or similar) within 7 days from today.
- Return all documents (or copies of) that arrived with the product (if they’re available – should be invoice, delivery note and datasheet)
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- Ship to the following address:

Attn: Returns ORDER REF:
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Please be advised that a 20% restock fee will apply to this order. The most up-to-date information regarding your order, invoice, and credit note status is available online in your Abcam account information. If your order was placed by credit card, your refund will be issued to your credit card. If your order was placed by Purchase Order, all customers must submit a credit note with their invoice to their Accounts Payable department to be handled. The Accounts Payable must have a copy of the credit note so the original invoice can be updated to reflect the correct amount owed.

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Abcam Scientific Support

Answered on Feb 09 2012

Application

Western blot

Sample

Human Cell lysate - whole cell (Glioblastoma neurospheres cells (primary))

Loading amount

30 µg

Specification

Glioblastoma neurospheres cells (primary)

Gel Running Conditions

Reduced Denaturing (12.5% gel)

Blocking step

Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 20°C

Abcam user community

Verified customer

Submitted Dec 06 2011

Application

Western blot

Sample

Human Cell lysate - whole cell (Platelets)

Loading amount

20 µg

Specification

Platelets

Treatment

Calcium Ionophore for 1 h

Gel Running Conditions

Reduced Denaturing (4-20%)

Blocking step

Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C

Dr. Mahesh Shivananjappa

Verified customer

Submitted Nov 14 2011

1-10 of 17 Abreviews or Q&A

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Key features and details

Overview

Product name

Description

Host species

Specificity

Tested applications

Species reactivity

Immunogen

Positive control

General notes

Properties

Form

Storage instructions

Storage buffer

Purity

Clonality

Clone number

Isotype

Research areas

Associated products

Alternative Versions

Compatible Secondaries

Isotype control

KO cell lysates

Recombinant Protein

Applications

Target

Function

Tissue specificity

Sequence similarities

Domain

Post-translationalmodifications

Cellular localization

Database links

Alternative names

Images

Protocols

Datasheets and documents

References (73)

Customer reviews and Q&As

Western blot abreview for Anti-Bad antibody [Y208]

Western blot abreview for Anti-Bad antibody [Y208]

Western blot abreview for Anti-Bad antibody [Y208]

Immunocytochemistry abreview for Anti-Bad antibody [Y208]

Immunocytochemistry abreview for Anti-Bad antibody [Y208]

Western blot abreview for Anti-Bad antibody [Y208]

Western blot abreview for Anti-Bad antibody [Y208]

Post-translational
modifications