HEK 293 EXPRESSION SYSTEM
Checking for Protein Expression
When you have got a stable cell line, which contains your target gene, you have to check cells for expression of this gene.
Fallowing protocol is useful in case of secreted proteins, which have C-terminal Fc-tag, binding specifically to protein A.
Reagents:
Protein A-Sepharose (Amersham cat. # 17-1279-01)
HBS buffer
HBST buffer
Procedures:
*NB For each sample (1.5 ml) use 50 ml Protein A-Sepharose
Prewash sepharose once with equal volume dd H2O (H2O removes ethanol in which the Sepharose has been stored). Spin down for 1 min at max speed in microcentrifuge (14 000 rpm) and carefully aspirate the supernatant.
Wash with the same volume HBST. Centrifuge for 1 min at max speed
Carefully aspirate the supernatant (repeat washing twice)
Resuspend Protein A-Sepharose in the same volume HBST. For example, you have 150 ul beads-add 150 ul HBST.
Take 1.5 ml of media, place in a microfuge tube, and mix with 50 ml Protein A-Sepharose in HBST. Protein A Sepharose should be transferred to the tube with a cut pipette tip which prevents the sepharose beads being damaged.
Put the tubes on a rocker for 2 h at 4oC. The longer the incubation time the better.
Centrifuge for 1 min at max speed in microcentrifuge (14 000 rpm) and aspirate the media
Add 1 ml HBST and rock the tubes gently until the beads get mixed with the solution
Centrifuge for 1 min at max speed (14 000 rpm) and aspirate the supernatant
Rinse with 1 ml HBS, spin it again and aspirate the supernatant.
Add 30 ml 1x sample buffer for SDS-PAGE to the beads
Boil for 3 min at 95oC. Flick the tube couple of times while boiling so the beads get mixed well with the sample buffer and the protein gets extracted from them to the solution.
Spin down for a minute at 14 000 r/m
Carefully take supernatant with loading tip and load it on a SDS-PAA gel (% depends of the MW of the protein)
Run for 1 h at 200V.
A background band at ~60 kDa will be present-so keep this in mind if your protein is ~ this size.
Western Blot
If your construct (target gene) does not contain any tags an antibody against your favorite protein might be used.
Materials:
Antibody (AB) against target protein, which has been bound with chromophore, enzyme and etc., to produce specific signal
Whatmanâ 3M chromatography paper
Nylon or nitrocellulose transfer membrane
Blocking agent (e. g. fat free milk powder, 4% solution, prepared before using)
Western transfer buffer
TBST buffer
Procedure:
*NB For this procedure the protein expression should be checked in a medium without FBS so the proteins in the serum does not interfere.
Mix 30 ml from media with 10 ml 4x Sample buffer. Boil the samples for 5 min at 95 C. Load them on a SDS-PAGE gel and run electrophoresis for ~1 h at 200V.
Assembling the transfer cells: On the bottom of a plastic box the positive electrode is placed. Place over it a plastic net and a sponge. Wet them with the Western transfer buffer. Two layers of whatman paper is placed on the sponge. Next place the membrane on top of the filter paper being careful not to introduce air bubbles between the filter paper and membrane. Next place the gel over the membrane and cover it again with two layers of Whatman paper, a sponge and a plastic net. Carefully roll out any bubbles with a small pipett. The transfer cell is then sealed with the negative electrode and the cover. The transfer cell is then righted vertically and filled with transfer buffer.
Run for 30 min. at 10V
Rinse the membrane with TBST
Block the membrane with 15 ml 4% fat free milk ( dissolve 2g of fat free milk in 50 ml of TBST). Incubate the membrane for 1h.
Rinse the membrane briefly with TBST
Incubate the membrane with the primary antibody against the protein ( the concentration of the antibody is determined according to the company standards and the antibody is diluted in TBST) for 2h or overnight (preferable) on a rocker at 4 C. A total volume of 15ml of the antibody solutions is sufficient to cover the membrane.
After the incubation, rinse the membrane 3 times for five to ten minutes each with TBST
If the primary antibody is not directly conjugated to the signal providing molecule(enzyme, chromophore etc.) a secondary antibody against the primary is needed. Dilute the secondary antibody in TBST according to the company instructions and incubate for ~ one hour.
Develop the signal by either adding an appropriate colored substrate for the enzyme or by measuring the induced light for the chromophore.
In case of using enzyme bound antibody the signal develops within 5-10 min.