shRNA Knockdown Validated by WB

WB-Validated ACAT2 Knockdown Cell Lysate Kit #L61849

基因名:
ACAT2
货号:
L61849

Information

Aliases:
ACAT2; Acetyl-CoA Acetyltransferase 2; Acetyl-CoA Acetyltransferase, Cytosolic; Acetyl-CoA Transferase-Like Protein; Cytosolic Acetoacetyl-CoA Thiolase; Acetoacetyl Coenzyme A Thiolase; EC 2.3.1.9; Acetyl-Coenzyme A Acetyltransferase 2 (Acetoacetyl Coenzyme A Thiolase); Acetyl-Coenzyme A Acetyltransferase 2; EC 2.3.1; ACTL


Background:
NCBI Gene Entry: 39


Storage
Stored at -20°C for 2 years.


Tested Cell Line
HeLa


Validation Methods
RT-qPCR; Western Blotting (WB)


Shipping
Shipped with gel ice packs. Immediately store the product in a standard freezer at -20°C upon receipt.


Instructions
This knockdown cell lysate should be paired with wild-type HeLa cell lysate for use. For Western blotting, we recommend running wild-type and knockdown lysates on the same gel, and loading each well with equal volume and equal amount of total proteins.


Manufacturing
The following protocol was used to generate mRNA knockdown cell lysate:
1.Release 0.5 million HeLa cells into a 35 mm tissue culture dish in 2 mL of the growth medium (DMEM containing 10% FBS and 1% pen/strep). Cell density should reach 50-60% confluence the following day.
2.24 h after cell release, pre-warm the shRNA lentiviral medium to 37°C.
3.Discard 1 mL of the original growth medium of the 35 mm dish.
4.Using a serological pipette, gently mix the lentiviral solution 3 times.
5.Carefully add 1 mL of the lentiviral solution to the well. Tip: To prevent splashing, add the solution to the dish along the wall.
6.Add a polybrene stock solution to the culture medium at a final concentration of 5 μg/mL. Gently swirl the dish to mix.
7.48 h after cell release, without discarding the original medium, add another 1 mL of lentiviral medium directly into the dish.
8.Add an additional polybrene stock solution into the dish to obtain a final concentration of 5 μg/mL. Tip: Now, the medium in the dish should be a total of 3 mL.
9.72 h after cell release, cells may reach confluence. Trypsinize the cells off the 35 mm dish and culture those cells in a 60 mm dish.
10.Add puromycin to the dish at a final concentration of 4 μg/mL. Tip: To assess the efficacy of puromycin selection, culture a dish of wild-type HeLa cells as a negative control.
11.Allow puromycin selection for 48 h. Almost all wild-type HeLa cells should die, while the dish infected with lentiviruses should have some remaining cells.
12.Replace the medium with regular growth medium without puromycin and allow the cells to grow to confluence before harvesting or staining.
13.Cells were lysed with IntactProtein™ cell/tissue lysis kit (Cat#415) and stored in -20℃.

Note: This product is for research use only.


Product Datasheet
For research use only

BETTER ROOTS BETTER FRUITS

WB-Validated ACAT2 Knockdown Cell Lysate Kit #L61849
shRNA Knockdown Validated by WB

ALIASES

ACAT2; Acetyl-CoA Acetyltransferase 2; Acetyl-CoA Acetyltransferase, Cytosolic; Acetyl-CoA Transferase-Like Protein; Cytosolic Acetoacetyl-CoA Thiolase; Acetoacetyl Coenzyme A Thiolase; EC 2.3.1.9; Acetyl-Coenzyme A Acetyltransferase 2 (Acetoacetyl Coenzyme A Thiolase); Acetyl-Coenzyme A Acetyltransferase 2; EC 2.3.1; ACTL


Background

NCBI Gene Entry: 39


TESTED CELL LINE

HeLa


VALIDATION METHODS

RT-qPCR; Western Blotting (WB)


SHIPPING

Shipped with gel ice packs. Immediately store the product in a standard freezer at -20°C upon receipt.


STORAGE

Stored at -20°C for 2 years.


INSTRUCTIONS FOR USE

This knockdown cell lysate should be paired with wild-type HeLa cell lysate for use. For Western blotting, we recommend running wild-type and knockdown lysates on the same gel, and loading each well with equal volume and equal amount of total proteins.


MANUFACTURING PROCESS

The following protocol was used to generate mRNA knockdown cell lysate:
1.Release 0.5 million HeLa cells into a 35 mm tissue culture dish in 2 mL of the growth medium (DMEM containing 10% FBS and 1% pen/strep). Cell density should reach 50-60% confluence the following day.
2.24 h after cell release, pre-warm the shRNA lentiviral medium to 37°C.
3.Discard 1 mL of the original growth medium of the 35 mm dish.
4.Using a serological pipette, gently mix the lentiviral solution 3 times.
5.Carefully add 1 mL of the lentiviral solution to the well. Tip: To prevent splashing, add the solution to the dish along the wall.
6.Add a polybrene stock solution to the culture medium at a final concentration of 5 μg/mL. Gently swirl the dish to mix.
7.48 h after cell release, without discarding the original medium, add another 1 mL of lentiviral medium directly into the dish.
8.Add an additional polybrene stock solution into the dish to obtain a final concentration of 5 μg/mL. Tip: Now, the medium in the dish should be a total of 3 mL.
9.72 h after cell release, cells may reach confluence. Trypsinize the cells off the 35 mm dish and culture those cells in a 60 mm dish.
10.Add puromycin to the dish at a final concentration of 4 μg/mL. Tip: To assess the efficacy of puromycin selection, culture a dish of wild-type HeLa cells as a negative control.
11.Allow puromycin selection for 48 h. Almost all wild-type HeLa cells should die, while the dish infected with lentiviruses should have some remaining cells.
12.Replace the medium with regular growth medium without puromycin and allow the cells to grow to confluence before harvesting or staining.
13.Cells were lysed with IntactProtein™ cell/tissue lysis kit (Cat#415) and stored in -20℃.

Note: This product is for research use only.









VALIDATION DATA


                                    


RT-qPCR analysis. HeLa cells were infected with ACAT2-specific shRNA lentiviral particles, total RNA was extracted from wild-type and knockdown cells, RT-qPCR was performed using gene-specific primers. ∆Ct (CtKD-CtWT) was used to calculate mRNA reduction (%) between wild-type and knockdown cells using the following formula: (1-1/2∆Ct) x 100%.



Western blotting analysis. ACAT2 protein expression in wild-type (WT) and shRNA knockdown (KD) HeLa cells was detected using Western blotting. Hsp90 α served as a loading control. The blots were incubated with primary antibodies (Cat#61849, 1:5,000) against ACAT2 and  Hsp90 α, respectively, followed by incubating with HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:20,000). Images were developed using FeQ™ ECL Substrate Kit (Cat#226).



RT-qPCR analysis. HeLa cells were infected with ACAT2-specific shRNA lentiviral particles, total RNA was extracted from wild-type and knockdown cells, RT-qPCR was performed using gene-specific primers. ∆Ct (CtKD-CtWT) was used to calculate mRNA reduction (%) between wild-type and knockdown cells using the following formula: (1-1/2∆Ct) x 100%.



Western blotting analysis. ACAT2 protein expression in wild-type (WT) and shRNA knockdown (KD) HeLa cells was detected using Western blotting. Hsp90 α served as a loading control. The blots were incubated with primary antibodies (Cat#61849, 1:5,000) against ACAT2 and  Hsp90 α, respectively, followed by incubating with HRP-conjugated goat anti-rabbit secondary antibody (Cat#201, 1:20,000). Images were developed using FeQ™ ECL Substrate Kit (Cat#226).


Material Safety Data Sheet (MSDS)

I. Identification:

Catalog Number: L61849

Product Name: WB-Validated ACAT2 Knockdown Cell Lysate Kit #L61849

Manufacturer & Supplier: Hefei SciBen Biotech Co., Ltd

Address:Building 6, 98 Jiulong Road, Hefei, Anhui 230601, China

Tel: 540.605.9777

Fax:540.605.9771

E-mail: info@genuinbiotech.com

II. Composition/Information on Ingredients:

Mixtures: No components need to be disclosed according to the applicable regulations.

III. Hazard Identification:

Classification of the substance or mixture: Not a hazardous substance or mixture.

GHS Label elements, including precautionary statements: Not a hazardous substance or mixture.

Hazards not otherwise classified (HNOC): Data not available.

IV. First Aid Measures:

IF exposed or concerned: Get medical advice.

IF in eyes:Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do.

If eye irritation persists: Get medical advice.

IF on skin: Wash with plenty of water and soap.

If skin irritation or rash occurs: Get medical advice.

Ingestion: Rinse mouth. Never give anything by mouth to an unconscious person. Do not induce vomiting. Call a physician.

V. Fire Fighting Measures:

Flash Point: Data not available.

Autoignition Temperature: Data not available.

Fire Extinguishing Media:Water spray, dry chemical, foam, or carbon dioxide.

Firefighting:Wear protective clothing and self-contained breathing apparatus to prevent contact with skin and eyes.

VI. Accidental Release Measures:

Absorb liquid with an absorbent material. Transfer contaminated absorbent to a chemical waste container for disposal.

VII. Handling And Storage:

Avoid inhalation and contact with eyes and skin. Avoid prolonged or repeated exposure. Store at –20 °C in tightly closed container.

VIII. Exposure Controls/Personal Protection:

Engineering Controls:Maintain adequate ventilation, eye wash and quick-drench facilities

IX. Physical and Chemical Properties:

Physical State:liquid.

Odor:Odorless.

Boiling Point:Data not available.

Melting Point:Data not available.

Volatile Organic Compound:Data not available.

Solubility in water:Readily miscible in water.

X. Stability and Reactivity:

Reactivity: Data not available

Chemical stability:Stable under recommended storage conditions.

Possibility of hazardous reactions: Data not available.

Conditions to avoid: Data not available.

Incompatible materials:Data not available.

Hazardous decomposition products: Data not available.

XI. Toxicological Information:

Acute toxicity: Data not available.

Inhalation: Data not available.

Dermal: Data not available.

Skin corrosion/irritation: Data not available.

Serious eye damage/eye irritation: Data not available.

Respiratory or skin sensitization: Data not available.

Reproductive toxicity: Data not available.

Aspiration hazard: Data not available.

XII. Ecological Information:

Toxicity: Harmful to aquatic life with long lasting effects.

Persistence and degradability:Data not available.

Bioaccumulative potential: Data not available.

XIII. Disposal Considerations:

Dispose of in accordance with federal, state and local environmental regulations.

XIV. Transport Information:

D.O.T.: This substance is considered non-hazardous for transport.

IATA: This substance is considered non-hazardous for air transport.

XV. Regulatory Information:

EU Regulation/Classification/Labeling Information: Not available for this product.

Chemical Inventory Status:

SARA Listed Component: None.

TSCA Listed Component: None.

Canada (WHMIS): DSL No, NDSL No.

XVI. Other Information:

This product is sold only for research use by personnel familiar with chemicals and who are well trained in good laboratory habits, such as avoiding spills, keeping hands clean at all times and not rubbing eyes with hands while working in the laboratory.

This product is sold only in microliter quantities for use in life sciences research. No other use is intended, and any other use may involve substantive hazards.

The above information is believed to be correct but does not purport to be all inclusive and shall be used only as a guide for experienced personnel. Hefei SciBen Biotech Co., Ltd shall not be held liable for any damage resulting from the handling of or from contact with the above product. The burden of safe use of this material rests entirely with the user.


Issuing Date: 8/16/2024 Version: 1.0