RFP antibody

Catalog No. ABIN967350

This anti-RFP antibody is a Mouse Monoclonal antibody detecting RFP in WB. Suitable for Discosoma. Independently validated for use in Western Blotting. This Primary Antibody has been cited in 1 publication.

Quick Overview for RFP antibody (ABIN967350)

Target: RFP (Red Fluorescent Protein (RFP))

Reactivity: Discosoma

Host: Mouse

Clonality: Monoclonal

Conjugate: This RFP antibody is un-conjugated

Application: Western Blotting (WB)

Clone: E64-1077

Product Details anti-RFP Antibody

Brand: BD Pharmingen™

Characteristics: 1. Since applications vary, each investigator should titrate the reagent to obtain optimal results.
2. Please refer to us for technical protocols.
3. Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
4. Source of all serum proteins is from USDA inspected abattoirs located in the United States.

Purification: The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.

Immunogen: DsRed1 recombinant protein

Isotype: IgG1

Application Details

Application Notes: Applications include western blot analysis (0.25-1.0 µg/ml). DsRed2 control lysate is provided as a positive control (component 51-16616N) and approximately 10 µl is recommended to visualize DsRed by western blot analysis.

Comment:

Related Products: ABIN967389

Restrictions: For Research Use only

Independent Validation (WB)

Validation #101231 (Western Blotting)

by: Institut für Virologie, Medizinische Hochschule Hannover

No.: #101231

Date: 06/19/2017

Antigen: DsRed

Lot Number: 6223801

Method validated: Western Blotting

Positive Control: Human foreskin fibroblasts infected with DsRed expressing virus

Negative Control: Cells infected with EGFP expressing virus

Notes: Passed. ABIN967350 specifically recognizes DsRed in extracts of cells expressing DsRed and GFP.

Validation Images

Immunoblot of DsRed or EGFP expressing human fibroblast lysates using ABIN967350. Cells were infected with EGFP-expressing virus (V1-gfp), DsRed-expressing viruses (V2-/V3-DsRed), or co-infected as indicated. The lane on the far right contains a sample from non-infected cells.

Full Methods

Primary Antibody: ABIN967350

Secondary Antibody: goat anti-mouse antibody (Pierce, 32230, lot QC212446)

Full Protocol:

• Grow fibroblasts in Dulbecco’s modified Eagle’s medium (Biochrom, F-0435, lot 0315F) supplemented with 10% fetal calf serum (FBS Good Forte, PAN Biotech, P40-47500, lot P130912)and 100U/ml penicillin, 100µg/ml streptomycin sulfate (PAN Biotech, P06-19100, lot 7530716) at 37°C and 5% CO₂ in 2ml on a tissue culture 6-well plate to 90% confluency.
• Infect cells at an MOI of 3 with
• • an EGFP-expressing virus V1-gfp,
  • a DsRed expressing viruse V2-DsRed,
  • a DsRed expressing viruse V3-DsRed,
  • virus V1-gfp and V2-DsRed, or
  • virus V1-gfp and V3-DsRed
• Grow cells for 4d at 37°C and 5% CO₂.
• Collect 8.5x10⁴ cells directly in 200µl of1x Laemmli SDS sample buffer, and boil them for 5min at 95°C.
• Separate the equivalent of 5000 cells of each sample on an Any kD SDS-PAGE gel (Bio-Rad, 456-9036, control 400100025) in a Bio-Rad Mini-Protean gel electrophoresis chamber for 45min at 250V and 21°C.
• Transfer proteins onto nitrocellulose membrane (GE Healthcare, 10600003, lot G7724136) with a TE77X semi-dry blotting system (SERVA) for 30min at 0.5A.
• Block the membrane using Roti-Block solution (Carl Roth, A151.1, lot 382190165) for 1h at RT.
• Incubate the membrane with primary
• • mouse anti-Red Fluorescent Protein (DsRed) antibody (antibodies-online, ABIN967350, lot 6223801)
  • rabbit anti-GFP (D5.1) XP (Cell Signalling, 2956, lot 4) antibody
• diluted 1:1000 in Roti-Block solution ON at RT.
• Wash the membrane 4x for 5min in PBS + 0.1% Tween20 (PBST).
• Incubate the membrane with secondary HRP-conjugated
• • goat anti-mouseantibody (Pierce, 32230, lot QC212446)
  • goat anti-rabbitantibody (Pierce, 32260, lot QH219992)
• diluted 1:2000 in PBST for 1h at RT.
• Wash the membrane 4x for 5min in PBST.
• Visualize protein bands using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, 34096, lot RH236205) on a LAS 3000 imager (Fuji).
• Strip the membrane using Restore PLUS Western blot stripping buffer (Thermo Scientific, 46428, lot PA196499) for 20min at RT.
• Wash the membrane 4x for 5min in PBST, and block for 1h at RT with Roti-Block solution.
• Incubate the membrane with primary rabbit anti-GAPDH antibody (Cell signalling, 2118, lot 10) diluted 1:2000 in PBST for 1h at RT.
• Wash the membrane 4x for 5min in PBST.
• Incubate the membrane with secondary goat anti-rabbit antibody (PIERCE, 32260, lot QH219992) diluted 1:2000 in PBST for 1h at RT.
• Wash the membrane 4x for 5min in PBST.
• Visualize protein bands using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific, 34096, lot RH236205) on a LAS 3000 imager (Fuji).

Experimental Notes:

The Red Fluorescent Protein (DsRed) antibody ABIN967350 reveals a protein of the expected molecular weight (31kDa) in lysates of cells infected with DsRed or with DsRed and EGFP expressing virus. A slightly lower MW band is visible in the lysates of EGFP (expected MW 28kDa) expressing cells. This might reflect some cross-reactivity of the DsRed antibody with the structurally similar EGFP. However, the overall selectivity of ABIN967350 towards DsRed is very good.

Handling

Format: Liquid

Concentration: 0.25 mg/mL

Buffer: Aqueous buffered solution containing BSA, glycerol, and ≤0.09 % sodium azide.

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: -20 °C

Storage Comment: Store undiluted at -20°C.

References for anti-RFP antibody (ABIN967350)

Wall, Socolich, Ranganathan: "The structural basis for red fluorescence in the tetrameric GFP homolog DsRed." in: Nature structural biology, Vol. 7, Issue 12, pp. 1133-8, (2000) (PubMed).

Target Details for RFP

Target: RFP (Red Fluorescent Protein (RFP))

Alternative Name: DsRed Fluorescent Protein

Target Type: Chemical

Background: DsRed is naturally occurring fluorescent protein from a coral of the genus Discosoma. DsRed, like green fluorescent protein (GFP) can be introduced into either cultured cells or in transgenic animals to study the localization of a specific protein. It can also be used in fusion constructs with a protein of interest to examine protein-protein interactions or as a general marker. The crystal structure of DsRed has been solved and shown that DsRed monomers exhibit similar topology to GFP, but have additional chemical modifications which account for differences in the spectra, when compared to GFP. DsRed has a longer emission/absorption wavelength than GFP. DsRed migrates at 31 kDa in SDS/PAGE. The antibody recognizes three forms of DsRed, 1, 2 and pTimer. A recombinant protein representing DsRed1 was used as the immunogen.

Molecular Weight: 31 kDa