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product information
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| background |
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His-Tag is a polyhistidine tag which consists of 6 histidine residues introduced on N- or C-terminus of the protein. The polyhistidine-tag can be used for recombinant protein detection using specific antibodies. |
| immunogen |
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KLH-conjugated synthetic peptide 6xHis |
| antibody format |
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mouse |
monoclonal |
IgG2b, clone HIS.H8 / EH158 |
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lyophilized |
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| quantity |
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100 µg |
for reconstitution add 100 µl, of sterile water. |
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| storage |
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store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. For storage at 4°C for several days to weeks, sodium azide to final 0.1 % can be added. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes. |
| tested applications |
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western blot (WB), dot blot (Dot), ELISA (ELISA), immunoprecipiation (IP), immunlocalization (IL) |
| related products |
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collection of antibodies to various tags |
| additional information |
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working dilution for ELISA, IL and IP needs to be determined experimentally. |
application information
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| recommended dilution |
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1 : 1000 with standard ECL (WB) |
| expected | apparent MW |
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n.a. |
| confirmed reactivity |
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6xHis
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| predicted reactivity |
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6xHis |
| not reactive in |
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no confirmed exceptions from predicted reactivity are currently known |
| additional information |
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antibody is present in 10 mM PBS, pH 7.2 |
| selected references |
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to be added when available, antibody available since July 2011. |
application example 
500 femtomoles of His-tagged proteins IsiA, NifH, PsbA, PsbB and PetC were loaded per gel well in Agrisera PEB extraction buffer. Proteins were separated on 4-12 % NuPAGE PAGE Bis-Tris polycacrylamide gel (Invitrogen) and blotted 1h to PVDF. Blots were blocked with for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (goat, anti-mouse IgG horse radish peroxidase conjugated, from Agrisera AS11 1772) diluted to 1:25 000 in 2 % ECL Advance blocking reagent for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL according to the manufacturers instructions. Exposure time was 5 seconds. Apparent molecular weight of recombinant proteins: IsiA - 27 kda, NifH - 34 kDa, PsbA - 30-37 kDa, PsbB - 40 kDa, PetC - 23 kDa. 
20 µl of media form Pichia Pastoris culter overexpressed His-Tegged proteins were separated on 12 % SDS-PAGE and blotted 1h to PVDF. Blots were blocked with 5% skim milk for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 1 000 for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in secondary antibody (goat anti-rabbit IgG horse radish peroxidase conjugated, from Bio-Rad) diluted to 1:15 000 in for 1h at RT with agitation. The blot was washed as above and developed for 5 min with ECL (GE Heltcare) according to the manufacturer’s instructions. Exposure time was 15 seconds.
M- protein ladder (Fermentas)
2- 20ul of medium before induction (PcGCE protein)
3-20ul of medium before induction (PcGCE S217N protein)
4-20ul of medium after 48h of induction (PcGCE protein)
5-20ul of medium after 48h of induction (PcGCE S217N protein)
6-20ul of medium after 96h of induction (PcGCE protein)
7-20ul of medium after 96h of induction (PcGCE S217N protein) Courtesy of Dr. Marta Derba-Maceluch, UPSC, Umeå
||| For applications or usage on species others than stated above Agrisera offers a payment-after-testing option. To learn more about this or for any questions on this product, please use the LiveChat option in the left menue bar or contact us at support@agrisera.com
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