Human ATG4A knockout HeLa cell lysate (ab257846)
Overview
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Product name
Human ATG4A knockout HeLa cell lysate -
Product overview
Knockout cell lysate achieved by CRISPR/Cas9. -
Parental Cell Line
HeLa -
Organism
Human -
Mutation description
Knockout achieved by using CRISPR/Cas9, 1 bp deletion in exon4. -
Passage number
<20 -
Knockout validation
Sanger Sequencing, Western Blot (WB) -
Reconstitution notes
To use as WB control, resuspend the lyophilizate in 50 µL of LDS* Sample Buffer to have a final concentration of 2 mg/ml. For reducing conditions, we recommend a final concentration of 0.1 M DTT.*Usage of SDS sample buffer is not recommended with these lyophilized lysates.
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Notes
Lysate preparation: Our lysates are made using RIPA buffer to which we add a protease inhibitor cocktail and phosphatase inhibitor cocktail (ratio: 300:100:10). This means that the protein of interest is denatured. If you require a native form of the protein please use the live cell version - found here. Please refer to our lysis protocol for further details on how our lysates are prepared.
User storage instructions: Lyophilizate may be stored at 4°C. After reconstitution, store at -20°C for short-term storage or -80°C for long-term storage.
Access thousands of knockout cell lysates, generated from commonly used cancer cell lines.
See here for more information on knockout cell lysates.Abcam has not and does not intend to apply for the REACH Authorisation of customers’ uses of products that contain European Authorisation list (Annex XIV) substances.
It is the responsibility of our customers to check the necessity of application of REACH Authorisation, and any other relevant authorisations, for their intended uses.This product is subject to limited use licenses from The Broad Institute and ERS Genomics Limited, and is developed with patented technology. For full details of the limited use licenses and relevant patents please refer to our limited use license and patent pages.
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Tested applications
Suitable for: WBmore details
Properties
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Storage instructions
Store at -80°C. Please refer to protocols. -
Components 1 kit ab260367 - Human ATG4A knockout HeLa cell lysate 1 x 100µg ab255929 - Human wild-type HeLa cell lysate 1 x 100µg -
Research areas
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Cell type
epithelial -
Disease
Adenocarcinoma -
Gender
Female -
STR Analysis
Amelogenin X D5S818: 11, 12 D13S317: 12, 13.3 D7S820: 8, 12 D16S539: 9, 10 vWA: 16, 18 TH01: 7 TPOX: 8,12 CSF1PO: 9, 10
Target
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Function
Cysteine protease required for autophagy, which cleaves the C-terminal part of either MAP1LC3, GABARAPL2 or GABARAP, allowing the liberation of form I. A subpopulation of form I is subsequently converted to a smaller form (form II). Form II, with a revealed C-terminal glycine, is considered to be the phosphatidylethanolamine (PE)-conjugated form, and has the capacity for the binding to autophagosomes. Preferred substrate is GABARAPL2 followed by MAP1LC3A and GABARAP. -
Tissue specificity
Widely expressed, at a low level, and the highest expression is observed in skeletal muscle and brain. Also detected in fetal liver. -
Sequence similarities
Belongs to the peptidase C54 family. -
Cellular localization
Cytoplasm. - Information by UniProt
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Alternative names
- AI627006
- APG4 autophagy 4 homolog A
- Apg4a
see all
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab257846 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. Predicted molecular weight: 45 kDa.
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| Notes |
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WB
Use at an assay dependent concentration. Predicted molecular weight: 45 kDa. |
Images
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Western blot - Human ATG4A knockout HeLa cell lysate (ab257846)
Lane 1: Wild-type HeLa cell lysate 20 μg
Lane 2: ATG4A knockout HeLa cell lysate 20 μg
Lane 3: HepG2 cell lysate 20 μg
Lane 4: Daudi cell lysate 20 μg
Lanes 1 - 4:Merged signal (red and green). Green - ab108322 observed at 45 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab108322 was shown to react with ATG4A in wild-type HeLa cells in Western blot with loss of signal observed in ATG4A knockout cell line ab265738 (ATG4A knockout cell lysate ab257846). Wild-type HeLa and ATG4A knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab108322 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging. -
Western blot - Human ATG4A knockout HeLa cell lysate (ab257846)Lane 1: Wild-type HeLa cell lysate 20 μg
Lane 2: ATG4A knockout HeLa cell lysate 20 μg
Lane 3: HepG2 cell lysate 20 μg
Lane 4: Daudi cell lysate 20 μg
Lanes 1 - 4:Merged signal (red and green). Green - ab223374 observed at 45 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.
ab223374 was shown to react with ATG4A in wild-type cells in Western blot with loss of signal observed in ATG4A knockout cell line ab265738 (ATG4A knockout cell lysate ab257846). Wild-type and ATG4A knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween®) before incubation with ab223374 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging. -
Sanger Sequencing - Human ATG4A knockout HeLa cell lysate (ab257846)Homozygous: 1 bp deletion in exon4
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.
Datasheets and documents
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SDS download
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Datasheet download
References (0)
ab257846 has not yet been referenced specifically in any publications.