E. coli Positive Control (Escherichia coli ) Whole Cell Lysate - expressing 6X His tag protein (ab2431)

E coli was grown in LB-Amp medium.

The E. coli was cultured using a standard protocol and harvested by centrifugation. The pellet was resuspended in 50mM Tris, 10mM EDTA, pH8.0, treated with ultrasonic sound, centrifuged and the supernatant was finally filtered (0.22µm).

Je peux vous confirmer que les cellules E. coli du produit ab2431 ont été préparé dans un tampon contenant 50mM Tris, 10mM EDTA, pH 8.0 et lysé par sonification ensuite. Le lysat était ensuite aliquoté et lyophilisé.

Thank you for your inquiry. We use the following 4X sample buffer: 4 mL mQ H2O 1 mL 0.5M Tris-Cl, pH 6.8 0.8 mL Glycerol 1.6 mL 10% SDS (w/v) 0.2 mL Bromphenol blue + 400µl Mercaptoethanol (for reduced gels) If you reconstitute the lysate directly in sample buffer we would recommend to use 1X. The total protein lysate is suggested to use as a standard or positive control when expressing 6H-tagged proteins in E. coli. When analyzing the total protein lysate on a gel by Western blotting a prominent band of 17 kDa will interact with anti-his-tag antibodies. The His-tagged protein is monomeric and will be detected under reducing and non-reducing conditions. It is recommended to use 5 mg – 20 mg protein lysate per lane for SDS-PAGE. After the lyophilized pellet is dissolved in 250 µl SDS-gel sample buffer (1 mg/ml protein lysate) it can be used direct for sample loading and then stored at –20°C. The sample can be reused several times..

The E. coli lysate containing the his-tag protein is a whole bacterial cell pellet lysate. The amount of bands detected with a his-tag antibody depends on the quality of the his-tag antibody and is therefore antibody dependent.

To our knowledge, this antibody has yet to be tested in this application. All tested applications are specified on Abcam product datasheets. If you decide to go ahead and purchase this product, please let us know how you get on and in return we will forward a reward of your choice, typically an Amazon gift voucher.