Anti-Parkin antibody (ab15954)

WB OK
IP: no band
293 cells
input lane: band seen
supernat.lane: band seen
IP band: no band seen
used 2 ug Ab per 500 ug protein (conc 1 mg/ml), incubation o/n 4 degree
(1/250)
protein A+G beads 2h 4 degree
elution: loading buffer

suggested to titrate Ab (e.g. 1/100 - 1/1000) to find optimal ratio for pull down
IP protocol?

ANSWER:

Thank you for your patience.

I have heard back from the lab with the following IP protocol:

Protocol for Immunoprecipitation ofparkin:

Cells cultured in 10 cm dishes were lysed on ice in cold lysis buffer containing the following: 1% Triton X-100, 10mM Tris pH 7.6, 50mM NaCl, 30 mM sodium pyrophosphate, 50 mM NaF, 5 mM EDTA, and 0.1 mM Na3VO4 for 20 min.

Lysates were centrifuged at 4°C at 16,000xg and supernatant fractions (about 100-500 µg equal amounts of total protein) were incubated with 4-8 µg rabbit polyclonal antibody against parkin for 1 hour at 4°C, followed by incubation with 50 µl of protein A/G plus agaroseat the same condition.



(Incubation can be done overnight depending on the amount of protein and affinity properties of the antibody at 4°C)

Immunoprecipitates were washed three times with the lysis buffer, then boiled in SDS loading buffer for 5 min and separated on SDS-polyacrylamide gel.

Half of the immunoprecipitates were for Western blot with anti α-tubulin (loading control), and the other half for Western blot with anti parkin.

Western blots were carried out using the ECL method according to the manufacturer’s protocol.

For experiments using rat brain homogenates, one whole brain was homogenized in 15 ml of ice-cold lysis buffer on ice in a tissue grinder. The homogenate was centrifuged at 16,000xg for 20 min and ultracentrifuged at 338,000xg for 30 min at 4°C. The supernatant fraction was used in immunoprecipitation or Western blot as described above.



I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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WB OK
IP: no band
293 cells
input lane: band seen
supernat.lane: band seen
IP band: no band seen
used 2 ug Ab per 500 ug protein (conc 1 mg/ml), incubation o/n 4 degree
(1/250)
protein A+G beads 2h 4 degree
elution: loading buffer

suggested to titrate Ab (e.g. 1/100 - 1/1000) to find optimal ratio for pull down
IP protocol?

ANSWER:

Thank you for contacting us.

I have heard back from the lab with the following advice and information:

The problem of the customer is HEK293 cells, which express very little parkin. Rat or mouse brain homogenate (freshly made) should be used as positive controls. Or one can use SH-SY5Y cells. If HEK293 cells are transfected with parkin, IP could be done very easily.It is better to use 5 ug of antibody and 1-4 mg of total cellular proteins.

I have again requestedan IP protocol in order to better assist you, and will let you know when I hear back with more information.

I hope this information is of some help so far. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
http://www.abcam.com/abreviews

Ab77924 is not showing a band in Western blot and ab15954 gives a band in knockdown samples.

ANSWER:

Thank you for your call today and for letting us know about the trouble with these antibodies.

As we discussed, I'm sending a free of charge vial of ab15954 on the order *** which should arrive tomorrow.

Please keep me updated about any further results with these antibodies, and if you have any data we would greatly appreciate seeing it. Let me know if you have any questions or if there is anything else that we can do for you, and I'll be happy to help you.

Recently, I examined Parkin protein with Anti-Parkin antibody (ab15954). The results showed that there were lots of unspecific bands but no bands were around the position for the size of parkin ( 52KD). I used HEK293 lysate for positive control. Loading control bands were good.
Since the advanced student bought the antibody 2 years ago, I doubt whether the product can be returned and exchanged for a new one? Or do you provide samples for testing parkin?

ANSWER:

I am sorry to hear that this antibody is not providing satisfactory results. It may be just that the antibody has gone through too many freeze-thaw cycles during use and may be leading to non-specific binding. Our Abpromise guarantee is for 6 months after purchase, so unfortunately we cannot replace or refund the product.

It is also possible that the antibody is detecting one of the isoforms of Parkin, as according to Swiss Prot there are at least 6 known isoforms.

http://www.uniprot.org/uniprot/O60260

We would recommend using brain, heart, testis or skeletal muscle as a positive control and loading at least 10 ug. We sell these products as ab29376 or ab30151.

http://www.abcam.com/Skeletal-Muscle-Rat-Tissue-Lysate-normal-tissue-ab29376.html

http://www.abcam.com/Brain-Mouse-Whole-Cell-Lysate-normal-tissue-ab30151.html

If you're seeing non-specific binding, you may want to try switching blocking buffers (i.e. use 5% BSA instead of milk), running a no primary control to see if the secondary is binding non-specifically, diluting the primary antibody more and incubating overnight at 4C instead of 1 hr at RT,and diluting the secondary more as well.

I hope this information helps. Please contact us with any other questions.