Overview

  • Product name

  • Description

    Rabbit polyclonal to GRAP
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, IHC-Pmore details
  • Species reactivity

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

    Synthetic peptide, corresponding to a region within amino acids 205-217 of Human GRAP (NP_006604).

  • Positive control

    • A549 whole cell lysate and U373 xenograft

Properties

Applications

Our Abpromise guarantee covers the use of ab97344 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 1/1000. Predicted molecular weight: 25 kDa.
IHC-P Use at an assay dependent concentration.

Target

Images

  • Anti-GRAP antibody (ab97344) at 1/1000 dilution + A549 whole cell lysate at 30 µg

    Predicted band size: 25 kDa



    12% SDS PAGE
  • ab97344 at 1/100 dilution staining GRAP in paraffin-embedded U373 xenograft by Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections).

References

ab97344 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

Answer

Thank you for your reply and for sending the additional information.

What dilution of the primary had you tried? We recommend 1:1000, but you may want to try diluting more. I'd also recommend switching the blocking buffer to 5% milk throughout the entire experiment as this is what was tested in the lab to generate the image on the datasheet. Also increase the initial blocking step from 30 mins to 1 - 2 hrs (even overnight at 4C).

You may also want to decrease the amount of protein that you're loading on the gel and increase the washes to 3 x 10 mins.

This is also the recommended cell lysis protocol that may help:

I. Monolayer Cells

The following steps should be performed on ice or at 4°C using pre-cold buffers. Remove culture medium and rinse a subconfluent, 100 mm cell culture plate with PBS twice.
Detach cells with a rubber policeman in 1 ml cold PBS and transfer cell suspension into a 1.5 ml microcentri-fuge tube.
Pellet cells by centrifuging at 3,000 rpm for 5 min. Remove the supernatant.
Suspend the pellet with 1.0 ml cold RIPA buffer (or other appropriate buffer) with freshly added (Protease Inhibi­tors) and/or (Phosphatase Inhibitors).
Allow the tube to stand on ice for 30 min, vortex every 10 min.
Centrifuge the resulting mixture at 14,000 x g for 15 min at 4°C. This separates the total protein (supernatant) from the cellular debris (pellet).
Transfer supernatant to a new tube for further analysis.
The cell lysate can be frozen at this point for long-term storage at -80°C.



II. Suspension Cells

Collect approximately 5.0 x 107 cells by low-speed centrifugation at RI for 5 min. Carefully remove culture medium.
Wash the pellet with PBS at RI, and collect by low-speed centrifugation. Carefully remove supernatant.
Add 1.0 ml of pre-cold RIPA buffer (or other appropriate buffer) with freshly added (Protease Inhibitors) and/ or (Phosphatase Inhibitors). Gently resuspend cells in RIPA buffer with a pipet and incubate on ice for 30 min.
Further disrupt and homogenize cells by passing through a 21-gauge needle, dounce homogenization or soni-cation, taking care not to raise the temperature of the lysate. (Optional: Add 10 pl of 10 mg/ml PMSF stock) Incubate 30 min on ice.

Transfer to microcentrifuge tube(s) and centrifuge at 10,000 x g for 10 min at 4°C. The supernatant fluid is the total cell lysate. Transfer the supernatant to a new microfuge tube and discard the pellet




I hope this information helps. If it does not improve the multiple band problem with ab97344 please let us know and we would be happy to replace or refund the product for you.

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