![Addgene/pAAV-hSyn-GRAB_NE1h/1unit/123309-AAVrg](images/Addgene/addgene-plasmid-123309-sequence-240668-map.png)
Ordering
Item | Catalog # | Description | Quantity | Price (USD) | ||
---|---|---|---|---|---|---|
Plasmid | 123309 | Standard format: Plasmid sent in bacteria as agar stab | 1 | $75 | Add to Cart | |
AAV9 | 123309-AAV9 | Virus (100 µL at titer ≥ 1×10¹³ vg/mL)and Plasmid.More Information | Add to Cart | |||
AAV Retrograde | 123309-AAVrg | Virus (100 µL at titer ≥ 7×10¹² vg/mL)and Plasmid.More Information | Add to Cart |
This material is available to academics and nonprofits only.
Backbone
- Vector backbonepAAV vector(Search Vector Database)
- Backbone sizew/o insert(bp)4495
- Vector typeAAV
Growth in Bacteria
- Bacterial Resistance(s)Ampicillin
- Growth Temperature37°C
- Growth Strain(s)NEB Stable
- Copy numberHigh Copy
Gene/Insert
- Gene/Insert nameGPCR activation based NE sensor GRAB_NE1h
- SpeciesH. sapiens (human)
- Insert Size (bp)1998
- PromoterhSyn
Cloning Information
- Cloning methodGibson Cloning
Resource Information
- Supplemental Documents
- pAAV-hsyn-GRAB-NE1h addgene.gb
- Terms and Licenses
- UBMTA
- Industry Terms
- Not Available to Industry
Depositor Comments
Please visit images/Addgene/449546.full.pdf for BioRxiv preprint.
Information for AAV9 (Catalog # 123309-AAV9)(Back to top)
Purpose
Ready-to-use AAV9 particles produced from pAAV-hSyn-GRAB_NE1h (#123309). In addition to the viral particles, you will also receive purified pAAV-hSyn-GRAB_NE1h plasmid DNA.
hSyn-driven expression of GRAB-NE1h norepinephrine sensor in neurons. These AAV preparations are suitable purity for injection into animals.Delivery
- Volume100 µL
- Titer≥ 1×10¹³ vg/mL
- Pricing$350 USD for preparation of 100 µL virus + $30 USD for plasmid.
- StorageStore at -80℃. Thaw just before use and keep on ice.
- ShipmentViral particles are shipped frozen on dry ice. Plasmid DNA (≥ 200ng) will also be included in the shipment.
Viral Production & Use
- Packaging Plasmidsencode adenoviral helper sequences and AAV rep gene, AAV9 cap gene
- BufferPBS + 0.001% Pluronic F-68
- SerotypeAAV9
- PurificationIodixanol gradient ultracentrifugation
Biosafety
Requestor is responsible for compliance withtheir institution"s biosafety regulations.Lentivirus is generally considered BSL-2. AAV isgenerally considered BSL-1, but may requireBSL-2 handling depending on the insert.Biosafety Guide
Resource Information
- Terms and Licenses
- Ancillary Agreement for Penn Vectors
- Terms of Use for Viral Vectors
- Industry Terms
- Not Available to Industry
Viral Quality Control
- Addgene ensures high quality viral vectors by optimizing and standardizing production protocols and performing rigorous quality control (QC) (see a list of our QC assays). Thespecific QC assays performed varies for each viral lot. To learn which specific QC assays were performed on your lot, please contact us.
- Titer: the exact titer of your sample will be reported on the tube. The titer you see listed on this page is the guaranteed minimum titer. See how titers are measured.
Visit our viral production page for moreinformation.
Information for AAV Retrograde (Catalog # 123309-AAVrg)(Back to top)
Purpose
Ready-to-use AAV Retrograde particles produced from pAAV-hSyn-GRAB_NE1h (#123309). In addition to the viral particles, you will also receive purified pAAV-hSyn-GRAB_NE1h plasmid DNA.
hSyn-driven expression of GRAB-NE1h norepinephrine sensor in neurons. These AAV were produced with a retrograde serotype, which permits retrograde access to projection neurons. These AAV preparations are suitable purity for injection into animals.Delivery
- Volume100 µL
- Titer≥ 7×10¹² vg/mL
- Pricing$350 USD for preparation of 100 µL virus + $30 USD for plasmid.
- StorageStore at -80℃. Thaw just before use and keep on ice.
- ShipmentViral particles are shipped frozen on dry ice. Plasmid DNA (≥ 200ng) will also be included in the shipment.
Viral Production & Use
- Packaging Plasmidsencode adenoviral helper sequences and AAV rep gene, AAV retrograde cap gene from rAAV2-retro helper (plasmid #81070)
- BufferPBS + 0.001% Pluronic F-68 + 200 mM NaCl
- SerotypeAAV retrograde (AAVrg)
- PurificationIodixanol gradient ultracentrifugation
Biosafety
Requestor is responsible for compliance withtheir institution"s biosafety regulations.Lentivirus is generally considered BSL-2. AAV isgenerally considered BSL-1, but may requireBSL-2 handling depending on the insert.Biosafety Guide
Resource Information
- Terms and Licenses
- Terms of Use for Viral Vectors
- Industry Terms
- Not Available to Industry
Viral Quality Control
- Addgene ensures high quality viral vectors by optimizing and standardizing production protocols and performing rigorous quality control (QC) (see a list of our QC assays). Thespecific QC assays performed varies for each viral lot. To learn which specific QC assays were performed on your lot, please contact us.
- Titer: the exact titer of your sample will be reported on the tube. The titer you see listed on this page is the guaranteed minimum titer. See how titers are measured.
Visit our viral production page for moreinformation.
Addgene Comments
Retrograde functionality is dependent on high viral titers. Addgene recommends not diluting your AAV preps prior to use.ebiomall.com
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1-5号蛋白浓度分别是(ug/ul):0.71.12.11.64.8
1-3号为一种细胞,4和5号为LO2细胞
1-4号采用同一种方法提取蛋白,5号采用的是另一种方法
问题:4号比5号的蛋白浓度高很多,但是条带还没没5号的明显??
4号和5号为什么会有拖尾尤其是4号,而1-3号则是条带太少(只有3条)
有多少种不同类型的震荡仪,型号是什么?
谢谢!
Western、IP 等。主要成分为50mM Tris (pH7.4),150mM NaCl,1% NP-40, 0.1% SDS。
使用本品裂解得到的蛋白样品,可以用BCA 蛋白浓度测定试剂盒测定蛋白浓度。由于含有
较高浓度的去垢剂,不能用Bradford 法测定由本裂解液裂解得到样品的蛋白浓度。
保存条件:4℃
制备细胞裂解产物:
1、800g 4℃离心5 分钟,收集细胞,估计细胞离心后的体积(PCV,106 cells ≈ 20ul PCV,
107 cells ≈ 100 ul PCV)
2、每50~100ul PCV 加入5 倍体积的蛋白裂解液(250~500ul),冰浴中放置10 分钟,且每隔5
分钟在漩涡混合仪震荡30 秒;
3、12000g 4℃离心10 分钟,将上清转移到新的离心管中,即得细胞总蛋白产物;
4、假如所得蛋白产物较为粘稠,可95℃加热5 分钟,然后迅速冰浴5 分钟,12000g 4℃离心
10 分钟,将上清转移到新的离心管中,即得细胞总蛋白产物。
制备组织裂解产物:
1、取50-100mg 组织在冰上剪成碎片,用预冷的PBS 洗涤2 次,离心弃去PBS;
2、加入0.5-1ml 预冷的蛋白裂解液;
3、4℃用玻璃匀浆器匀浆20-40 次,直到95%的细胞被破碎,然后在冰浴中放置10 分钟,且每
隔5 分钟在漩涡混合仪震荡30 秒;
4、12000g 4℃离心10 分钟,将上清转移到新的离心管中,即得组织总蛋白产物;
5、假如所得蛋白产物较为粘稠,可95℃加热5 分钟,然后迅速冰浴5 分钟,12000g 4℃离心
10 分钟,将上清转移到新的离心管中,即得组织总蛋白产物。
6、20%的甘油保存于-70℃或-20℃。
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