![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.](http://images.novusbio.com/fullsize/HeLa-Chloroquine-Treated---Untreated-Cell-Lysate-Western-Blot-NBP2-49689-img0005.jpg "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.")
| Reactivity | HuSpecies Glossary |
| Applications | WB, PAGE |
| Concentration | 1.0 mg/ml |
| Description | 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. |
| Marker | Autophagy/LC3 Positive Control |
| 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. |
| Dilutions |
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| 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). |
| Storage | Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles. |
| Buffer | 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 |
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The Role of LC3 within the Autophagic Pathway We at Novus Biologicals have a broad antibody database covering the area of autophagy - over 1400 reagents in total. Autophagy is the bulk degradation of cytoplasmic components - literally, self-digestion of the cell. Double-membrane vesicles, called ... Read full blog post. |
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![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.](http://images.novusbio.com/fullsize/HeLa-Chloroquine-Treated---Untreated-Cell-Lysate-Western-Blot-NBP2-49689-img0002.jpg "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] - 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.](http://images.novusbio.com/fullsize/HeLa-Chloroquine-Treated---Untreated-Cell-Lysate-Western-Blot-NBP2-49689-img0003.jpg "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] - 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.](http://images.novusbio.com/fullsize/HeLa-Chloroquine-Treated---Untreated-Cell-Lysate-Western-Blot-NBP2-49689-img0004.jpg "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.")