How do you lyse cells for a Western blot?

Suspension Cells

  1. Pipet cells into a fresh conical tube and place on ice.
  2. Spin cells on low speed at 4°C, and aspirate off media.
  3. Add 10 ml ice cold PBS, and gently invert tube to wash cells.
  4. Spin cells on low speed, and aspirate off supernatant.
  5. Repeat wash and aspiration.

How do you split cells in 96 well plate?

Splitting 96-well master plates

  1. Feed cells 2 hours before splitting, and have new 96-well plates ready with 100 ul of ES media/well.
  2. Aspirate off media and wash cells with PBS.
  3. Add 30ul of trypsin (0.05%) and incubate for 5 min.
  4. Using multichannel pipettor, add 100 ul of ES media and titerate 5-10X.

How many cells do you harvest for Western blot?

A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 – 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer.

How do you lysis a cell?

The technique involves freezing a cell suspension in a dry ice/ethanol bath or freezer and then thawing the material at room temperature or 37°C. This method of lysis causes cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing.

How many cells does a 6-well plate have?

Useful information for various sizes of cell culture dishes and flasks

Catalog No. Cells at confluency*
6-well 140675 1.2 x 106
12-well 150628 0.5 x 106
24-well 142475 0.24 x 106

How much protein is sufficient for western blot?

Protein samples for western blotting can be soluble protein fluids, cell/tissue lysates or immunoprecipitated proteins. The protein loading differs from different samples, basically, the recommended protein loading of purified protein is no more than 100 ng, and the loading of cell/tissue lysate could be 10-40 μg.

How do you store cells in a Western blot?

Blots, once dried, can be kept in sealed containers, sitting on paper tissue, in a refrigerator for long periods of time before probing with primary antibody.

How to grow ES cells in a 96 well plate?

1. Grow ES cells in a 96-well plate to be over-confluent. 2. Quickly invert the plate over to dump media and remove excess liquid by blotting on paper towels. 3. Wash cells 1x with 150 uL PBS, then dump PBS. 4. Add 50 ul of Bradley Lysis Buffer containing proteinase K. 5. Replace lid and seal the plate with parafilm.

How to prepare a western blot for cell lysis?

Transfer to a microfuge tube and clarify the lysate by spinning at 4°C, for 10 minutes at 12,000 RPM. Decant the supernatant to a fresh tube, and discard cell pellet. Store on ice for immediate use, or at -20°C or -80°C until needed. Measure protein concentration with BCA, Lowry, or Bradford assays, or by absorbance, prior to loading on a gel.

How to prepare a tissue culture dish for lysis?

Keep tissue culture dish on ice throughout. Add appropriate volume ice cold lysis buffer (with fresh protease inhibitors), to the flask, approximately 1ml for a 100 mm tissue culture dish. (Alternatively, cells can be removed from the flask with Trypsin-EDTA and then prepared using the suspension cell instructions below).

How to prepare suspension cells for cell lysis?

Suspension cells: Aspirate off liquid. Gently resuspend the cell pellet in ice cold cell lysis buffer (with fresh protease inhibitors), use 1 ml buffer for 107 cells. Incubate cells for 30 minutes on ice. If needed, sonicate the lysates on ice for 15-30 seconds to disrupt genomic DNA and cellular components.