Expi293 cells were cultured in a 1:1 mixture of Expi293:Freestyle media (Thermofisher). At a cell density of ~2.5x106 cells/ml, 250 ml of cells were transfected using 200 μl FectoPRO (Polyplus Transfection) reagent mixed with 100 μg PRMT5 and 100 μg HA-tagged WDR77 plasmids13 . One day after transfection, 3 mM valproic acid and 0.4% w/v glucose were added to the culture. Two days after transfection, cells were split into multiple flasks with 30 ml cells each and treated with compound or DMSO at a final 0.2% DMSO concentration. Cell sample was collected after 6 hours, pelleted and washed 3 times with ice cold PBS. Additional aliquots were analyzed for viability and cell number using a Vi-Cell XR Cell Counter (Beckman Coulter) and all samples were determined to have >98% viability based on trypan blue exclusion. Each sample was lysed in buffer 50 mM HEPES pH 7.5, 300 mM NaCl, 1 mM TCEP, 1% v/v Tween-20 and 2 mM reduced glutathione. The PRMT5:WDR77 complex was immunoprecipitated by anti-HA agarose resin (Thermofisher) and then eluted by 50 μM 3X-HA peptide (AnaSpec). Each eluate was measured by intact mass LC-MS to determine the percentage of complex with compound adduct.
Anti ha agarose resin
Manufactured by Thermo Fisher Scientific
The Anti-HA agarose resin is a chromatography resin designed for the purification of proteins tagged with the hemagglutinin (HA) epitope. It is composed of agarose beads covalently coupled with an anti-HA antibody, allowing for the specific capture and isolation of HA-tagged proteins from complex samples.
Automatically generated - may contain errors
3 protocols using anti ha agarose resin
1
Expi293 Transfection and PRMT5-WDR77 Immunoprecipitation
2
E. coli Gp46 Protein Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
E. coli BL21 (DE3) carrying gp46pET28b was cultured in Luria-Bertani (LB) medium supplemented with kanamycin (50 μg/mL) at 37°C overnight. The next day, the bacterial culture was diluted 1:100 in fresh LB medium with kanamycin and cultured until reaching OD600 nm = 0.4–0.8, subsequently isopropyl β-D-thiogalactoside (400 μM) was used to induce protein expression. After 4 h, the bacteria were pelleted, resuspended in 2 mL of TBS, and lysed using a French press (16,000 psi, three times). The clear lysate was applied onto an anti-HA agarose resin (Thermo Fisher Scientific, 26181) and incubated at 4°C overnight with rotation. After three washes with TBS supplemented with 0.05% Tween20, bound Gp46 protein was eluted using 3 M NaSCN. Buffer exchange was performed using Zeba Spin Desalting Columns (Thermo Fisher Scientific, 89882). The protein was stored in buffer containing 150 mM NaCl and 50 mM Tris at pH 7.5. To minimize the possibility of E. coli HU protein co-purification, anion exchange chromatography was performed using Macro-Prep DEAE Support (Bio-Rad, 1560020). The concentration of the protein samples was determined using a Qubit assay (Invitrogen, Q33211).
3
Purification of HU_HA Protein from F. tularensis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
F. tularensis subsp. holarctica FSC200 carrying the HU protein labeled with an HA tag (HU_HA) (Pavlik and Spidlova, 2022 (link)) was cultured overnight in Chamberlain’s medium supplemented with kanamycin (20 μg/mL). The pellet was resuspended in Tris-buffered saline (TBS) and lysed using a French press (16,000 psi, three times). The clear lysate was mixed with anti-HA agarose resin (Thermo Fisher Scientific, 26,181) and incubated at 4°C overnight with rotation. HU_HA proteins bound to the resin were washed three times with TBS supplemented with 0.05% Tween20 and eluted with 3 M NaSCN. Buffer exchange was performed using Zeba Spin Desalting Columns (Thermo Fisher Scientific, 89882). The protein was stored in buffer containing 150 mM NaCl and 50 mM Tris at pH 7.5. The concentration of the protein samples was determined using a Qubit assay (Invitrogen, Q33211).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!