Lentiviral shRNA validated by WB

WB-Validated APPL1 Lentiviral shRNA Knockdown Kit #V61661

基因名：
APPL1
货号：
V61661
RT-qPCR analysis. HeLa cells were infected with APPL1-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. APPL1 protein expression in wild-type (WT) and shRNA knockdown (KD) HeLa cells was detected using Western blotting. β-Tubulin served as a loading control. The blots were incubated with primary antibodies (Cat#61664, 1:5,000) against APPL1 and β-Tubulin, 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 APPL1-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. APPL1 protein expression in wild-type (WT) and shRNA knockdown (KD) HeLa cells was detected using Western blotting. β-Tubulin served as a loading control. The blots were incubated with primary antibodies (Cat#61664, 1:5,000) against APPL1 and β-Tubulin, 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).

Information

Aliases:
APPL1; Adaptor Protein, Phosphotyrosine Interacting With PH Domain And Leucine Zipper 1; APPL; DCC-Interacting Protein 13-Alpha; Adaptor Protein, Phosphotyrosine Interaction, PH Domain And Leucine Zipper Containing 1; Adapter Protein Containing PH Domain, PTB Domain And Leucine Zipper Motif 1; Dip13-Alpha; Adaptor Protein Containing PH Domain, PTB Domain And Leucine Zipper Motif 13; Signaling Adaptor Protein DIP13alpha; AKT2 Interactor; DIP13alpha; KIAA1428; MODY14; DIP13A


Background:
NCBI Gene Entry: 26060


Kit Components
 1 . WB-Validated APPL1 shRNA lentiviral particles (4 mL)

 2 . Non-Target shRNA lentiviral particles (4 mL)

 3. Verification Tools:

  (1) WB-Validated APPL1 knockdown cell lysate (25 μg)

  (2) Wild type cell lysate (25 μg)

  (3) KD-Validated anti-APPL1 rabbit monoclonal ab (5 μL)


Storage
1. Lentiviral particles, -80 °C for 1 year.
2. Cell lysate kit, -20 °C for 2 years.
3. Antibody, -20 °C for 1 year.


Tested Cell Line
HeLa


Validation Methods
RT-qPCR; Western Blotting (WB)


Shipping
Shipped with dry ice. Immediately store the product in a standard freezer at -80°C upon receipt.


Instructions
The following protocol uses HeLa cell as an example assuming your cell culture medium is DMEM-based. 
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.


Note: 1. This product is for research use only. 

      2. This product is only supplied to end users.

      3. Do not use this product for commercial.


Size Price
4 mL ¥ 8000.00
Datasheet
MSDS

RECENTLY VIEWED

Note

Product Datasheet
For research use only

BETTER ROOTS BETTER FRUITS

WB-Validated APPL1 Lentiviral shRNA Knockdown Kit #V61661
Lentiviral shRNA validated by WB

ALIASES

APPL1; Adaptor Protein, Phosphotyrosine Interacting With PH Domain And Leucine Zipper 1; APPL; DCC-Interacting Protein 13-Alpha; Adaptor Protein, Phosphotyrosine Interaction, PH Domain And Leucine Zipper Containing 1; Adapter Protein Containing PH Domain, PTB Domain And Leucine Zipper Motif 1; Dip13-Alpha; Adaptor Protein Containing PH Domain, PTB Domain And Leucine Zipper Motif 13; Signaling Adaptor Protein DIP13alpha; AKT2 Interactor; DIP13alpha; KIAA1428; MODY14; DIP13A


Background

NCBI Gene Entry: 26060



TESTED CELL LINE

HeLa


VALIDATION METHODS

RT-qPCR; Western Blotting (WB)


SHIPPING

Shipped with dry ice. Immediately store the product in a standard freezer at -80°C upon receipt.


STORAGE

1. Lentiviral particles, -80 °C for 1 year. 2. Cell lysate kit, -20 °C for 2 years. 3. Antibody, -20 °C for 1 year.


INSTRUCTIONS FOR USE

The following protocol uses HeLa cell as an example assuming your cell culture medium is DMEM-based. 
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.


Note: 1. This product is for research use only. 

      2. This product is only supplied to end users.

      3. Do not use this product for commercial.









VALIDATION DATA


                                    

                                


                            

                            

                                        

                                            

                                                
                                            

                                            


                                            
RT-qPCR analysis. HeLa cells were infected with APPL1-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. APPL1 protein expression in wild-type (WT) and shRNA knockdown (KD) HeLa cells was detected using Western blotting. β-Tubulin served as a loading control. The blots were incubated with primary antibodies (Cat#61664, 1:5,000) against APPL1 and β-Tubulin, 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 APPL1-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. APPL1 protein expression in wild-type (WT) and shRNA knockdown (KD) HeLa cells was detected using Western blotting. β-Tubulin served as a loading control. The blots were incubated with primary antibodies (Cat#61664, 1:5,000) against APPL1 and β-Tubulin, 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).

                                        


                            

                            
                        

                    

                


    


    

                

                    

                        
GenuIN Biotech LLC

                        
2000 Kraft Drive, Suite 1110, Blacksburg, VA 24060, USA 

                        
Tel: 0551-163635518  Fax: 540.605.9771

                        
E-mail: info@genuinbiotech.com  www.genuinbiotech.com

                    

                    
Material Safety Data Sheet (MSDS)

                    

                        
I. Identification:

                        
Catalog Number: V61661

                        
Product Name:  WB-Validated APPL1 Lentiviral shRNA Knockdown Kit #V61661

                        
 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:support@scibenbio.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) or not covered by GHS Biohazard, Biosafety Level 2:   Lentiviruses are a subset of retroviruses, able to integrate into host chromosomes, and to infect nondividing cells. More recent generation vectors have been designed to diminish possible recombination resulting in a wild type- potentially infectious virus. It is usually recommended that work with nonhuman lentiviruses that are incapable of establishing productive infections in humans be conducted at BSL-2.






                        
 IV. First Aid Measures:

                        
Inhalation:  If breathed in, move person into fresh air. If not breathing, give artificial respiration. 

                        
In case of skin contact:Wash off with soap and plenty of water.

                        
In case of eye contact:Flush eyes with water as a precaution.

                        
If swallowed: Never give anything by mouth to an unconscious person. Rinse mouth with water.



                        
 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:Strong oxidizing agents.  

                        
Hazardous decomposition products: Other 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:  Data not available.

                        
Persistence and degradability:Data not available.

                        
Bioaccumulative potential: Data not available.

                        
Mobility in soil: 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



                    

                    
GenuIN Biotech LLC provides the information contained herein in good faith but makes no representation as to its comprehensiveness or accuracy. This document is intended only as a guide to the appropriate precautionary handling of the material by a properly trained person using this product. Individuals receiving the information must exercise their independent judgment in determining its appropriateness for a particular purpose. GenuIN Biotech LLC makes no representations or warranties, either express or implied, including without limitation any warranties of merchantability, fitness for a particular purpose with respect to the information set forth herein or the product to which the information refers. Accordingly, GenuIN Biotech LLC will not be responsible for damages resulting from use of or reliance upon this information.