Human Perforin 1 (PRF1) Protein (Active)

Human Perforin 1 (PRF1) Protein (Active) is an active recombinant Human protein produced in E. coli using Prokaryotic expression, for use in cell culture and activity assays.

Target	Perforin 1 (PRF1)
Origin	Human
Expression	Recombinant
Tested Applications	ELISA, WB, SDS-PAGE
Host	E. coli
Conjugation	Unconjugated
Form	Lyophilized
Purity	> 95%
Reconstitution	To keep the original salt concentration, we recommend reconstituting to the original concentration prior to lyophilization (see Concentration) in ddH₂O. If a lower concentration is required, dilute in 20 mM Tris, 150 mM NaCl, pH 8.0. If a higher concentration is required, the product can be reconstituted directly in 20 mM Tris, 150 mM NaCl, pH 8.0, though please note that this will change the overall salt concentration. The stock concentration should be between 0.1-1.0 mg/ml. Do not vortex.
Storage	Store at 2-8 °C for up to one month. Store at -80 °C for up to one year. Avoid repeated freeze/thaw cycles.
UniProt Primary AC	P14222 (UniProt, ExPASy)
UniProt Secondary AC	B2R6X4, Q59F57, Q86WX7
UniProt Entry Name	PERF_HUMAN
KEGG	hsa:5551
String	9606.ENSP00000398568
Molecular Weight	Calculated MW: 61.5 kDa Observed MW (SDS-PAGE): 62 kDa
Sequence Fragment	Lys32-Phe316
Sequence	KRSHKFVPG AWLAGEGVDV TSLRRSGSFP VDTQRFLRPD GTCTLCENAL QEGTLQRLPL ALTNWRAQGS GCQRHVTRAK VSSTEAVARD AARSIRNDWK VGLDVTPKPT SNVHVSVAGS HSQAANFAAQ KTHQDQYSFS TDTVECRFYS FHVVHTPPLH PDFKRALGDL PHHFNASTQP AYLRLISNYG THFIRAVELG GRISALTALR TCELALEGLT DNEVEDCLTV EAQVNIGIHG SISAEAKACE EKKKKHKMTA SFHQTYRERH SEVVGGHHTS INDLLF
Tag	N-terminal His tag and GST tag
Buffer	Prior to lyophilization: 20 mM Tris, 150 mM NaCl, pH 8.0, containing 0.01% Sarcosyl and 5% Trehalose.
Activity	Active
Biological Activity	Perforin 1 (PRF1) binds to cell plasma membranes and oligomerises in a Ca²⁺-dependent manner to form pores in the target cell. Calreticulin (CRT), a protein that binds Ca²⁺ ions, has been identified as an interactor of PRF1, therefore a binding ELISA assay was conducted to detect the interaction of recombinant human PRF1 and recombinant human CRT. Briefly, PRF1 was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 µl were then transferred to CRT-coated microplate wells and incubated for 2 h at 37 °C. Wells were washed with PBST and incubated for 1 h with anti-PRF1 polyclonal antibody, then aspirated and washed 3 times. After incubation with HRP-conjugated secondary antibody, the wells were aspirated and washed 3 times. TMB substrate solution was added to the wells, which were then incubated for 15-25 minutes at 37 °C. Finally, 50 µl stop solution was added to the wells and the absorbance was measured at 450 nm immediately. The binding activity of PRF1 and CRT is shown in Figure 4, where it can be seen that the effect is dose-dependent. The activity of recombinant Human PRF1 Protein was observed with a hemolysis assay measuring lysis of rabbit erythrocytes (RaE) and compared with a negative control (Figures 5 and 6). Briefly, recombinant Human PRF1 Protein was diluted 2-fold in 0.9% NaCl. To each well, 50 µl of diluted PRF1 (12.5 µg/ml) and 10 µl 0.1 M CaCl₂ was added, followed by 50 µl 0.25% RaE. The wells were mixed gently and the plate was incubated for 20 h at 37 °C, 5% CO₂. For the negative control, 10 µl 0.9% NaCl was used instead of 10 µl 0.1 M CaCl₂. The minimal effective concentration of PRF1 was observed as 12.5 μg/ml.
Endotoxin Level	< 1.0 EU per 1 µg (LAL method)
Concentration	Prior to lyophilization: 220 µg/ml
Availability	Shipped within 5-7 working days.
Note	This product is for research use only. Not for human consumption, cosmetic, therapeutic or diagnostic use.

Human Perforin 1 (PRF1) Protein (Active)

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