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HeLa Ser/Thr Phosphatase-Inhibited Whole Cell Lysate Set: Calyculin-Treated and Vehicle-Treated (ab135395)

Submit a review Q&A (1)
Western Blot
  • SDS-PAGE

Overview

  • Product name

    HeLa Ser/Thr Phosphatase-Inhibited Whole Cell Lysate Set: Calyculin-Treated and Vehicle-Treated

  • Product overview

    HeLa whole cell extracts were collected in lysis buffer after 3 hour treatment with Calyculin A (50 nM) or vehicle (DMSO) in serum-free media. The protein content was determined by BCA assay. Calyculin A is a potent phosphatase inhibitor. Unlike Okadaic Acid, which only acts on protein phosphatase PP2A, Calyculin A inhibits both PP1 and PP2A. Cells treated with Calyculin A accumulate serine/threonine phosphorylation making them a strong positive control for phospho-threonine or phospho-serine specific antibodies.


    Concentration:


    HeLa Lysate - Vehicle Treated Control - 200 µg at 1 mg/mL
    HeLa Lysate - Calyculin Treated - 200 µg at 1 mg/mL


    The lysate is supplied in SDS-PAGE loading buffer with DTT and is ready to load on a gel.

  • Tested applications

    Suitable for: WB, SDS-PAGEmore details

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab135395 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
Use at an assay dependent concentration.
SDS-PAGE
Use at an assay dependent concentration.
Notes
WB
Use at an assay dependent concentration.
SDS-PAGE
Use at an assay dependent concentration.

Images

  • Western Blot
    Western Blot

    All lanes: Anti-Phosphoserine/Threonine antibody (ab17464) at 1/1000

    Lane 1: Marker
    Lane 2: HeLa Whole Cell Lysate - Calyculin Treated at 20 µg
    Lane 3: HeLa Whole Cell Lysate – Untreated Control at 20 µg


    Secondary:
    Goat polyclonal to Rabbit IgG – Pre-Adsorbed (HRP) at 1/6000 developed using the ECL technique.

    Performed under reducing conditions.

  • SDS-PAGE
    SDS-PAGE
    Lane 1: Marker
    Lane 2: HeLa Whole Cell Lysate - Calyculin Treated at 20 µg
    Lane 3: HeLa Whole Cell Lysate – Untreated control at 20 µg


    Gel
    10-20% acrylamide separation gel

Protocols

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

References (0)

Publishing research using ab135395? Please let us know so that we can cite the reference in this datasheet.

ab135395 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

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Question



We want to order this item (135395) as a control for some of our experiments. We are interested in the vehicle treated lysate only. However, I have a few technical questions before we place the order-

1. What is the lysis buffer that is used to make this extract? Is it a hypotonic lysis buffer, or do you use detergents (like Triton or Tween)?

2. Are the cell debris (nuclei, mitochondria) removed from this? And are the lipids also removed?

3. Do you have any information about the cell-density of the HeLa cells used prior to the lysis?

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Answer



1. What is the lysis buffer that is used to make this extract? Is it a hypotonic lysis buffer, or do you use detergents (like Triton or Tween)?


- I can confirm that for ab135395 the cells are directly lysated in SDS buffer. Since SDS generates a sticky lysate due to DNA content, the lysate is sonicated on ice to break the DNA. The formulation is 50mM Tris pH 6.8, 150mM NaCl, 8mM NaF, 1% SDS, 100mM DTT, plus protease inhibitor cocktail.



2. Are the cell debris (nuclei, mitochondria) removed from this? And are the lipids also removed?


- No lipids or cells debris is removed as this is a whole cell lysate.



3. Do you have any information about the cell-density of the HeLa cells used prior to the lysis?


- I can confirm that we generate the material from multiple plates that are about 95% confluent.



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