Atg4B Rabbit Recombinant mAb | Monoclonal Antibodies

Atg4B Rabbit Recombinant mAb

Catalog No.A5151

For research use only.

Filter:

  • WB

Usage Information

Application WB,ELISA
Dilution
WB
1:1000
Reactivity Human Mouse Rat
MW (kDa) 44kDa
Source Rabbit
Concentration 1mg/ml
Storage buffer 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide.
Storage Store at –20°C.

Protocol

WB

Western Blotting

Sample preparation

1. Aspirate media from cultures and Wash the cells with 1X PBS.
2. Lyse cells by adding 1X SDS sample buffer and transfer the extract to a microcentrifuge tube. Keep onice.
3. Sonicate for 10–15 sec to complete cell lysis and shear DNA.
4. Heat a 20 µl sample to 95–100°C for 5 min, then cool on ice.
5. Centrifuge for 5 min (with Microcentrifuge).
6. Load appropriate volumes of samples onto SDS-PAGE gel (loading quantity of protein sample depends on the concentration of extracted proteins).
NOTE: At the same time, please load the pre-stained molecular weight markers to determine molecular weights and verify electrotransfer.
7. Electrotransfer to nitrocellulose/PVDF membrane.

Membrane Blocking and Antibody Incubations

a. Blocking

1. (Optional) After transfer, wash the transferred membrane with TBS for 5 min at room temperature.
2. Incubate the membrane in the blocking buffer for 1 hr at room temperature.
3. Wash three times for 5 min each with TBST.

b. Antibodies Incubation

1. Incubate membrane and primary antibody (at the appropriate dilution and diluent recommended) in a primary antibody dilution buffer with gentle agitation overnight at 4°C.
2. Wash three times for 5 min each with TBST.
3. Incubate membrane with an appropriate second antibodydissolved in the blocking buffer with gentle agitation for 1 hr at room temperature.
4. Wash three times for 5 min each with TBST.
5. Proceed with detection.

Detection of Proteins

1. After antibodies incubation, Wash membrane three times for 5 minutes in TBST.
2. PrepareECL Reagent (or other chromogenic agents/substrate according to your second antibody). Mix well.
3. Incubate substrate with membrane for 1 minute, remove excess solution (membrane remains wet), wrap in plastic and expose to X-ray film.

Description

Specificity Atg4B Rabbit Recombinant mAb detects endogenous level of Atg4B.
Background Autophagy is an evolutionarily conserved catabolic pathway within all eukaryotic cells that plays a critical housekeeping function to remove senescent or damaged proteins and organelles through lysosome-mediated degradation. Autophagy also plays roles in generating substrates for maintaining cellular energy during times of nutrient insufficiency and hypoxia and in host defenses against intracellular pathogens. Atg4 plays dual roles in the progression of autophagy through the irreversible (proteolytic cleavage) and reversible (lipid deconjugation) modification of Atg8. ATG4B (autophagin-1) is an enzyme that is essential for autophagy in yeast and mammals. ATG4B (autophagin-1) has broad specificity for almost all of the mammalian Atg8 homologues and is the sole enzyme reported to efficiently cleave LC3 precursors and to deconjugate lipid from LC3-PE. Of the four autophagins identified (ATG4A, ATG4B, ATG4C, and ATG4D), ATG4B (autophagin-1) shows the highest catalytic efficiency for cleaving the C terminus of LC3B.

Datasheet & SDS

Tech Support

If you have any other enquiries, please leave a message.

* Indicates a Required Field

Please enter your name.
Please enter your email. Please enter a valid email address.
Please write something to us.