HeLa Chloroquine Treated / Untreated Cell Lysate

Novus Biologicals | Catalog # NBP2-49689

Novus Biologicals
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Key Product Details

Species

Human

Applications

SDS-PAGE, Western Blot

Product Summary for HeLa Chloroquine Treated / Untreated Cell Lysate

NBP2-49689 contains 1 vial each of 0.1ml of HeLa Chloroquine treated and 0.1ml of HeLa untreated cell lysate. This lysate set is useful as a positive and negative controls for targets such as LC3, p62 etc. and the researchers should use anti-Human primary antibodies when working with these lysates.

Product Specifications

Application Notes

HeLa Chloroquine treated / untreated whole cell lysates are provided as positive and negative control for Western blot analysis in Autophagy research. The lysates are provided as one vial of treated and one vial of untreated samples. Use 10 ul per lane for a standard mini-gel blot (approx. 1 mg/ml).

Marker

Autophagy/LC3 Positive Control

Type

Cell

Tissue

Cervix

Subcellular Fraction

Chloroquine Treated / Untreated

Scientific Data Images for HeLa Chloroquine Treated / Untreated Cell Lysate

Western Blot: HeLa Chloroquine Treated / Untreated Cell Lysate [NBP2-49689]

Western Blot: HeLa Chloroquine Treated / Untreated Cell Lysate [NBP2-49689]

Western Blot: HeLa Chloroquine Treated / Untreated Cell Lysate [NBP2-49689] - HeLa cells were treated with (+) or without 50 uM (-) of Chloriquine (CQ) for 24 hours. Total cell lysates were prepared and separated on a 12% gel by SDS-PAGE. Protein was transferred to PVDF membrane and blocked in 5% non-fat milk. The membrane was then probed with 2 ug/ml of p62/SQSTM1 antibody (NBP1-42821) in 1% milk and detected with an anti-rabbit HRP secondary antibody using chemiluminescence. Note the upregulation of p62 (arrowhead) in response to chloroquine treatment and the blockage of autophagy.
Western Blot: HeLa Chloroquine Treated / Untreated Cell Lysate [NBP2-49689]

Western Blot: HeLa Chloroquine Treated / Untreated Cell Lysate [NBP2-49689]

Western Blot: HeLa Chloroquine Treated / Untreated Cell Lysate [NBP2-49689] - 10 ug of Chloroquine treated (+) and untreated (-) HeLa lysates in 1x Laemmli sample buffer (NBP2-49689) were separated on a 4-15% gel by SDS-PAGE, transferred to 0.2 um PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 1 ug/ml monoclonal anti-LC3 (NBP2--46892) and detected with an anti-rabbit secondary antibody using chemiluminescence.
Western Blot: HeLa Chloroquine Treated / Untreated Cell Lysate [NBP2-49689]

Western Blot: HeLa Chloroquine Treated / Untreated Cell Lysate [NBP2-49689]

Western Blot: HeLa Chloroquine Treated / Untreated Cell Lysate [NBP2-49689] - Human Cervical carcinoma cells (HeLa) were treated with (+) and without (-) 50 uM Chloroquine overnight. Whole cell protein lysates were prepared in 1x Laemmli sample buffer and approximately 10 ug of each lysate (NBP2-49689) was separated on a 4-15% gel by SDS-PAGE, transferred to 0.2 um PVDF membrane and blocked in 5% non-fat milk in TBST. The membrane was probed with 1 ug/ml anti-LC3 (NB100-2220) and 1 ug/ml anti-alpha tubulin (NB100-690) as a loading control, and detected with the appropriate secondary antibodies using chemiluminescence.
Western Blot: HeLa Chloroquine Treated / Untreated Cell Lysate [NBP2-49689]

Western Blot: HeLa Chloroquine Treated / Untreated Cell Lysate [NBP2-49689]

Western Blot: HeLa Chloroquine Treated / Untreated Cell Lysate [NBP2-49689] - HeLa cells were treated with vehicle (-) or with 50 uM chloriquine (+) for 24 hours and the total cell lysates were prepared. The lysates were separated on a 12% gel by SDS-PAGE. Protein was transferred to PVDF mebbrane and blocked in 5% non-fat milk. The membrane was then probed with 1 ug/ml of p62/SQSTM1 antibody (NBP1-48320) in 1% milk and dectected with an anti-rabbit HRP secondary antibody using chemiluminescence. Note the upregulation of p62 (arrowhead) in response to chloroquine treatment and the blockage of autophagy.

Kit Contents for HeLa Chloroquine Treated / Untreated Cell Lysate

  • 0.1 ml HeLa Chloroquine treated whole cell lysate
  • 0.1 ml HeLa untreated whole cell lysate

Formulation, Preparation, and Storage

Preparation Method

HeLa cells were cultured under standard laboratory conditions until semi-confluent (70-80%). The cells were then treated with or without Chloroquine to 50 uM for 24 hours. The cells were washed in PBS and directly lysed into 1X Laemmli sample buffer containing BME. Each lysate is sonicated and boiled before being tested in Western blot for reactivity to LC3. Tubulin reactivity in each lysate is shown as a loading control.

Formulation

The protein lysate is prepared in 1x Laemmli sample buffer with BME. Boiling before loading is not necessary.

Preservative

No Preservative

Concentration

1.0 mg/ml

Shipping

The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.

Storage

Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.

Background: HeLa

Chloroquine (CQ) or N4-(7-Chloro-4-quinolinyl)-N1,N1-dimethyl-1,4-pentanediamine diphosphate salt (C18H26ClN3. 2H3PO4) is an anti-malarial drug which is well known for its inhibitory effects on apoptosis and autophagy in various cell lines. CQ is a weak base which can partition into the acidic vesicles such as endosomes and lysosomes, thereby, resulting in the inhibition of endosomal acidification and lysosomal enzyme activity. Lysosomotropic autophagy inhibitor CQ decreases the activity of lysosomal acid hydrolase enzymes which impairs the lysosomal function (lysosomal degradation of autophagosomes) ultimately resulting in the accumulation of autophagosomes. Because autophagosomal membranes are rich in lapidated form of LC3 protein (LC3-II), CQ treated cells are often exploited for their usefulness as positive controls in autophagy related research, especially for Western blot and ICC/IF analysis of LC3 of cultured cells. In ICC/IF, the CQ treated cells would be expected to show an enhanced number of autophagosomes compared to vehicle treated cells. Similarly, in WB analysis, the lysate of the CQ treated cells must show an enhanced LC3-II levels compared to the lysate of vehicle treated cells. HeLa Chloroquine Treated / Untreated Cell Lysate [NBP2-49689] is provided as a ready to use pair of lysates, 0.1ml of HeLa Chloroquine treated (positive control) and 0.1ml of HeLa untreated (negative control), for Western blot with anti-Human LC3 or p62 antibodies. NBP2-49689 has been validated using monoclonal as well as polyclonal anti-LC3 and antibodies, and the lysate of CQ treated cells depicted an increased level of LC3-II (see data image).

Alternate Names

LC3 Lysate, LC3B western blot

Additional HeLa Products

Product Documents for HeLa Chloroquine Treated / Untreated Cell Lysate

Certificate of Analysis

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Product Specific Notices for HeLa Chloroquine Treated / Untreated Cell Lysate

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

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